Norflurane

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

- dosing conducted on Days 6 through 15 of pregnancy

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Norflurane

EC Number:

212-377-0

EC Name:

Norflurane

Cas Number:

811-97-2

Molecular formula:

C2H2F4

IUPAC Name:

1,1,1,2-tetrafluoroethane

Details on test material:

1,1,1,2-tetrafluoroethane (HFC 134a) was supplied by Imperial Chemical Industries Limited, Mond Division, Runcorn, Cheshire, UK. Its purity was >99.5%.

Test animals

Species:

rat

Strain:

other: Alpk/APfSD Wistar-derived

Details on test animals or test system and environmental conditions:

Nulliparous female rats from the specific pathogen-free colony at Alderley Park, Cheshire, UK were supplied when approximately 10 weeks old and within a weigh range of 186 to 248g. They were mated overnight at the breeding unit, and the following morning vaginal smears from each female were examined for the presence of spermatozoa.

The presence of sperm was regarded as an indication of successful mating and the day on which sperm were detected was Day 0 of pregnancy. On this day the animals were delivered to the experimental unit. They arrived in two batches on two consecutive days and were supplied with a record of the record of the identity of the male with which each female was mated.

The rats were housed up to eight per cage in cages constructed of wire mesh (internal measurements length 42 cm x width 45 cm x height 19cm) which were divided by the same mesh so that four rats were housed in each side.
The cages were suspended over collecting trays lined with adsorbent paper and were arranged four on each of three levels within large long-term inhalation chambers (2m3 capacity).

All animals were given Alderley Park rat cubes supplied by Oakes Limited, Congleton, Cheshire, UK and tap water ad libitum except during dosing. A 12 hour light and 12 hour dark cycle (starting at 6.00 am) was maintained in the animal room where the large inhalation chambers were situated. The temperature and relative humidity within the chambers were controlled at approximately 21°C and 50% respectively. Measurements were not, however, as it had been found previously that these remained relatively constant for a given experiment.

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

Dosing by whole body exposure for 6 hours daily commenced on Day 6 (6 February 1978 for batch 1 females and 7 February 1978 for batch 2 females) and continued up to and including Day 15 of gestation. For the first test exposure on 6 Februaruy , the large inhalation chambers were used. Due to the accidental release of one cylinder of HFC 134a, smaller Perspex inhalation chambers (66 Litre capacity) had to be used from 7 February onwards. This enabled the experiment to continue with the remaining test material. The smaller chambers were situated in a laboratory adjacent to the animal room in which the rats were housed and therefore the rats had to be transported to and from this laboratory on each day of exposure. The atmospheres within both types of exposure chamber were generated by mixing known volumes of HFC 134a with clean dry air using rotameters as indicators. Atmospheric concentrations were monitored approximately 2-3 times and hour by gas liquid chromatography (GLC).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A Pye gas-liquid chromatography (GLC) system with a gas sampling valve was used to analyse 10 ml aliquots of the test atmosphere taken from the exposure chambers using gas-tight syringes. Samples were either withdrawn directly from the Perspex chambers or from one of four points from the large long-term inhalation chambers. Atmospheric concentrations were monitored approximately 2-3 times an hour.
An isothermal flame ionisation detector on the GLC was used to analyse the eleunt gas stream, and peak areas from the exposure chamber samples were compared with peak areas obtained from identical volume samples taken from the relevant cylinder of pre-diluted gas.

Details on mating procedure:

Females were mated overnight at the breeding unit, and the following morning vaginal smears from each female were examined for the presence of spermatozoa.
The presence of sperm was regarded as an indication of successful mating and the day on which sperm were detected was Day 0 of pregnancy. On this day the animals were delivered to the experimental unit. They arrived in two batches on two consecutive days and were supplied with a record of the record of the identity of the male with which each female was mated.

Duration of treatment / exposure:

from day 6 to day 15 of gestation

Frequency of treatment:

6 hours / day

Duration of test:

21 Days (on day 21 of pregnancy the rats were terminated).

No. of animals per sex per dose:

29 - 30 females / group

Control animals:

yes, concurrent no treatment

Details on study design:

The animals were allocated to four experimental groups by referring to the mating records to ensure that all the females mated with a given male were distributed as equally as possible amongst the groups. The animals were then ear-marked for identification purposes.

Examinations

Maternal examinations:

Clinical effects: The animals were observed daily throughout the experiment and both during and at the end of each exposure period they were observed for signs of distress. A more detailed examination was made when the animals were weighed and any abnormalities noted.

Bodyweights: Individual bodyweights were recorded on Days, 0, 6, 16 and 21 of pregnancy.

Parental Pathology: On Day 21 of pregnancy the rats were killed by cervical dislocation. An autopsy was carried out and tissues of all females were examined macroscopically. Lungs from ten pregnant females per group were submitted in formol corrosive for histopathological examination after perfusion with formol saline. They were trimmed, processed and embedded in paraffin wax. Sections 5um thick were cut and stained with haematoxylin and eosin. Heart, liver, kidneys, uterus, placenta, ovary, adrenals and lungs (not already fixed in formol corrosive) from the pregnant females from which foetuses were preserved were stored in fomol saline for possible future examination.

Ovaries and uterine content:

The ovaries were examined macroscopically and preserved in formol for possible future examination.
During autopsy the intact gravid uterus was removed and weighed. It was then examined for the number of live foetuses and resorptions. Resporptions were classified as early or late, being identified as the latter when foetal tissues were distinguishable. Corpora lutea were also counted and numbers recorded.

Fetal examinations:

The foetuses were assigned letters of the alphabet identifying their position in utero and were then removed in order starting at the proximal end of the left horn and ending at the proximal end of the right horn. The weight and sex of each individual foetus were recorded and at the same time viability was assessed using the following criteria – colour, breathing and movement. Any reduction in viability was noted.

Assessment of teratogenicity: After the external abnormalities including cleft palate had been recorded. Alternate foetuses (starting randomly within each litter) were fixed in 70% methanol. Approximately 24 hours later, they were eviscerated (the viscera being examined macroscopically for abnormalities) and the fat pads covering the cervical and thoracic vertebrae were removed. The internal sex was checked against that recorded externally.

Each foetues was returned to the methanol and then processed and stained with Alizarin Red using the method of Staples and Schnell for subsequent skeletal examination. During this examination, ossified bones were examined for abnormalities and for degree of ossification. The overall ossification of forelimb and hindlimb digits were assessed on a four point scale.

The remaining foetuses were fixed in Bouin’s fixative for decalcification. After 3-4 weeks fixation in Bouin’s the foetuses were stored in 70% methanol for subsequent soft tissue examination. The head and thorax of each foetus were serially sectioned using the technique of Wilson. The abdominal contents were examined by dissection and again the sex checked internally. The kidneys were sectioned transversely to examine their internal structure. Each of two prosectors examined approximately equal numbers from each group in an attempt to avoid operator bias.
A minimum of twenty litters per group were fixed and examined, excess litters being discarded.

Statistics:

Statistical analysis was carried out by comparing results from the HFC 134a exposed groups with results from the control group. A one way analysis of variance of maternal bodyweight gain, number of implants, viable foetuses, resorptions, mean (litter) foetal weight and total litter (live foetuses) weight was carried out. Group means were then compared by use of a one-sided Student’s t-test.

Foetal survival was considered by analysing numbers of live foetuses as a proportion of implantations after transformation using the double arcsine function of Freeman and Tukey. A one-way analysis of variance was also used to assess this variable.

Pre- and post implantation loss were calculated using the following equations:

Pre-implantation loss =

(no. of corpora lutea – no. of implantations) x 100 / no. of corpora lutea

Post-implantation loss =

(no. of implantations – no. of live foetuses) x 100 / no. of implantations

Skeletal anomalies and degree of ossification were assessed by the use of the 2x2 contingency tables of Finney et al. If clarification of a particular finding was necessary, litter mean scores were calculated. The means for each litter were then considered by one-way analysis of variance and the group mean scores compared by Student’s t-test based on the residual mean square.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Effect levels (maternal animals)

Dose descriptor:

NOEL

Effect level:

>= 50 000 ppm

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

1

Dose descriptor:

NOAEC

Effect level:

>= 50 000 ppm

Basis for effect level:

other: teratogenicity

2

Dose descriptor:

NOAEC

Effect level:

>= 50 000 ppm

Basis for effect level:

other: embryotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Atmospheric concentrations (Table 2)

Actual atmospheric concentrations were mostly within an acceptable range ±20% of the theoretical levels and of these most fell within 10%. Theoretical levels have been referred to throughout this summary.

Table 2: Mean Daily Atmospheric Concentrations of HFC 134a (ppm)

Day of Gestation		Day of Exposure	1000 ppm		10 000 ppm		50 000 ppm
Batch 1 Animals	Batch 2 Animals		Mean	S.D.	Mean	S.D.	Mean	S.D.
6	-	1	1280*	800	7300*	3200	33400*	8600
7	6	2	1040	210	9100	2900	48700	8600
8	7	3	1010	200	11900	2400	28500*	7300
9	8	4	1050	170	9300	1400	46500	5300
10	9	5	1040	250	12700*	3800	39600	7600
11	10	6	1080	280	10600	2300	51400	9800
12	11	7	1200	300	10800	2800	44200	7800
13	12	8	1020	100	9900	700	50900	2600
14	13	9	950	200	9900	800	51900	4900
15	14	10	1000	190	9400	2300	45000	6900
-	15	11	1050	180	10000	1100	57000	3500

* outside the range of ±20%

Maternal Clinical Observations

None of the animals showed an adverse reaction to exposure to HFC 134a at any of the dose levels administered. Apart from very slight lacerations around the heads of 8 animals in the 50 000 ppm group (numbers 97 – 104) and 16 animals in the 10 000 ppm group (numbers 65 – 80) caused by fighting, all the animals remained in good health for the duration of the experiment.

There were several animals which gained no weight during the first 6 days of the experiment but this was due to accidental deprivation of food and water for approximately 24 hours prior to them being weighed on Day 6. The animals affected were 121 – 126 50 000 ppm, 89 – 94 10 000 ppm, 57 – 62 1000 ppm, and 25 – 29 control group, but no adverse effects were seen subsequently.

Maternal bodyweights (Table 3)

There were no differences in mean maternal bodyweight gain between the control and those of animals exposed to HFC 134a at any of the dose levels administered (the statistical treatment is given in Table 6). Mean maternal bodyweights were also similar in all groups.

Table 3: Inter-group Comparison of Mean Maternal Bodyweights (g)

Dose Level HFC 134a ppm	Day of Pregnancy					Mean bodyweight Gains (g)
	0	6	10	16	21
0	221.7	251.4	270.0	299.5	353.0	131.3
(Control)	(15.0)	(24.7)	(20.3)	(27.2)	(26.2)	(16.8)
1000	221.7	248.9	271.3	300.7	354.5	132.5
	(12.4)	(21.9)	(16.3)	(21.6)	(20.8)	(15.2)
10 000	216.2	246.2	268.7	299.0	349.3	133.1
	(13.2)	(23.8)	(19.8)	(28.3)	(23.9)	(15.0)
50 000	222.7	252.3	270.4	296.3	350.0	127.3
	(13.3)	(21.1)	(14.7)	(19.3)	(17.4)	(10.4)

Standard deviations are in parenthesis

Maternal pathology (Table 4)

In a large proportion of the lungs examined microscopically there was blood in the alveoli, which was a consequence of death by cervical dislocation and probably also accounts for the ‘patchy’ lungs frequently seen at autopsy.

The presence of polymorphs either marginally disposed in the blood vessels and / or in the perivascular tissue was observed in some of the lung tissues examined. Sometimes but not always there was also a focal proliferation of the epithelial lining of the terminal bronchioles. This finding was present alone in some of the tissues examined.

When the severity of the lung lesion was considered, there appeared to be some correlation with dose level. However, the range of microscopic lung findings in not unusual in laboratory rats.

Table 4: Histological Examination of Maternal Lungs

Findings	Control	HFC 134a
		1000 ppm	10 000 ppm	50 000 ppm
Number examined	10	10	10	10
Polymorphs: few	4	3	5	3
Polymorphs: many	0	2	2	4
Number of lungs affected	4	5	7	7
Bronchiolar Irritation: slight	3	2	2	4
Bronchiolar Irritation: marked	0	0	2	4
Number of lungs affected	3	2	4	8

Litter data (Tables 5 and 6)

The statistical treatment of the litter data is shown in Table 6. There were no significant differences in the numbers of implantations , live foetuses and resorptions when control and HFC 134a data were statistically compared. However, the mean foetal weight was statistically significantly (p<0.05) lower in the 50 000 ppm group when compared to the controls although there was no reduction in mean litter weights. The mean gravid uterus weights were also similar in all groups. Pre- and post-implantation losses were unaffected by treatment and the percentage of male foetuses although variable, was within normal limits.

External foetal abnormalities

Three foetuses had external abnormalities, two in the control group and one the 50 000 ppm group. One control had agnathia, exophthalmia with open eyes and a proboscis, while the second had small ears. That in the 50 000 ppm group had and umbilical hernia.

Table 5: Inter-group Comparison of Litter Data

Dose Level of HFC 134a	Number of Females	Number of Pregnancies / Litters	Mean Number of Implantations per Litter	Resorptions (Numbers and %)		Mean Number of Corpora Lutea	Mean % pre-Implantation Loss	Mean % post-Implantation Loss
				Early	Late
0 (Control)	29	23	11.9	28 10.2	1 0.0	12.6	6.9	10.7
			(2.1)			(1.4)	(11.8)	(10.8)
1000	30	29	12.1	20 5.7	1 0.3	12.3	8.1	5.7
			(2.9)			(2.9)	(17.6)	(7.0)
10 000	30	26	12.6	28 8.6	4 1.2	12.1	4.7	10.1
			(1.6)			(2.8)	(9.5)	(9.2)
50 000	30	27	12.3	17 5.1	1 0.3	13.4	10.0	5.4
			(2.5)			(1.5)	(16.5)	(7.3)

Standard deviations are in parentheses

Table 5 (Continued): Inter-group Comparison of Litter Data

Dose Level of HFC 134a	Number of Live Foetuses	Number of Male Foetuses	Number of Female Foetuses	% of Male Foetuses	Mean Gravid Uterus Weight (g)	Mean Foetal Weight (g)	Mean Litter Weight (g)
0 Control	246	138	108	56.1	75.4	5.2	55.8
					(17.4)	(0.37)	(13.5)
1000	331	146	185	44.1	78.3	5.1	57.6
					(18.0)	(0.23)	(14.1)
10 000	295	136	159	46.1	79.2	5.2	58.6
					(12.6)	(0.36)	(9.5)
50 000	313	151	162	48.2	78.5	5.0*	57.5
					(15.0)	(0.33)	(11.8)

Standard deviations are in parentheses

* Statistically significant compared to the control group mean at the 5% level (Student’s t-test, one-sided)

Table 6: Statistical Treatment of Maternal and Litter Data

Parameter	Dose Level of HFC 134a (ppm)				Approximate 95% Confidence Limits^1
	0 (Control)	1000	10 000	50 000
Mean female weight gain (g)	131.3	132.7	133.1	127.3	±5.6
Number of pregnant females	23	29	26	27	-
Mean number of implants	11.9	12.1	12.6	12.3	±0.9
Mean number of live foetuses	10.7	11.4	11.3	11.6	±1.0
Mean number of resorptions	1.0	0.7	1.2	0.6	±0.4
Transformed live foetuses / implants	2.48	2.61	2.47	2.64	±0.11
Mean foetal weight (g)	5.2	5.1	5.2	5.0*	±0.12
Mean litter weight (g)	55.8	57.6	58.6	57.5	±4.8

1. Based on the mean group size

* Statistically significant compared to the control group mean at the 5% level (Student’s t-test, one-sided)

Skeletal Examination (Table 7)

There was some indication of retardation of ossification in the 50 000 ppm group foetuses as shown by the statistically significant effects on vertebrae, sternebrae, digits and calcaneum. There was also a significantly increased incidence of anomalies of the sternebrae (bipartite or misaligned) seen in this group. Occasional significant differences in ossification were seen in the 1000ppm group but none were seen at 10 000 ppm. When the ossification of cervical vertebrae centra and the digits was examined on a litter basis, only the ossification of the hind limb digits in 50 000 ppm group was significant.

Table 7: Summary of statistically significant effects on the vertebrae, sternebrae, digits and calcaneum

Description	Dose level of HFC 134a
	0 (Control)		1000		10 000		50 000
	No.	%	No.	%	No.	%	No.	%
Number of foetuses examined	126		130		125		126
VERTEBRAE
Cervical
Centra 1-2 not ossified	5	4.0	16	12.3*	4	3.2	15	11.9*
LUMBAR
Total number of vertebrae ossified
36 ossified	29	23.0	29	22.3	35	28.0	44	34.9*
39 ossified	11	8.7	3	2.4*	7	5.6	6	4.8
STERNEBRAE
Total partially ossified	65	51.6	65	50.0	53	42.4	89	70.6**
Total abnormal	6	4.8	6	4.6	5	4.0	16	12.7*
FORELIMB
Assessment of ossification of digits
Grade 0 (good)	63	50.0	51	39.2	60	48.0	42	33.3*
Grade –1	53	42.1	66	50.8	57	45.6	75	59.5**
HINDLIMB
Assessment of ossification of digits
Grade 0 (good)	41	32.5	36	27.7	33	26.4	25	19.8*
Grade –1	45	35.7	30	23.1*	40	32.0	31	24.6
Grade –2	36	28.6	59	45.4	51	40.8	65	51.6**
Calcaneum partially ossified	28	22.2	22	16.9	22	17.6	12	9.5**

* P <0.05 Statistically significant compared to the control group

** P <0.01 Statistically significant compared to the control group

Soft Tissue Examination (Table 8)

The highest incidence of any abnormality seen was pelvic dilatation of the kidneys. The incidence in all groups was low and unrelated to treatment.

Table 8: Inter-group Comparison of Soft Tissue Abnormalities

Description	Dose level of HFC 134a (ppm)
	0 (Control)	1000	10 000	50 000
Number of foetuses examined	120	130	124	129
KIDNEYS
Right slight pelvic dilatation	7	4	4	3
Left slight pelvic dilatation	3**	1	6	1
Both slight pelvic dilatation	4	1	2	1
Right moderate pelvic dilatation	1**
Both moderate pelvic dilatation		1
Number of foetuses affected	14	7	12	5
URETERS
Folded				1*
Dilated (hydroureter)				1*
BRAIN
Slight expansion of lateral ventricles	2
EYES
Left anophthalmia	1

* Same foetus

** Same foetus classified in both categories

Applicant's summary and conclusion

Conclusions:

HFC 134a was neither teratogenic nor embryotoxic at levels up to 50000 ppm, but at this level it may be slightly foetotoxic.

Executive summary:

Pregnant Alpk/APfSD Wistar-derived rats (29 - 30/group) were exposed (6 h/d) to 0, 1,000, 10,000 and 50,000 ppm (0, 4,170, 41,700, 208,000mg/m3) HFC-134a from day 6 to 15 of gestation. The exposure to HFC-134a produced abnormal clinical signs but did not affect the maternal body weights. Mean foetal weights were slightly but significantly lower in the offspring of rats exposed to 50,000 ppm. Embryonic and foetal survival were unaffected by the treatment. There was no evidence for teratogenicity but skeletal ossification was slightly retarded in the top dose (50,000 ppm). It was concluded that HFC-134a was neither teratogenic nor embryotoxic at levels up to 50,000 ppm, but at this highest level HFC-134a might be slightly foetotoxic.