Reaction products of 2-(chloromethyl)oxirane and glycerol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: well documented publication, which meets basic scientific principles

Data source

Reference

Reference Type:

publication

Title:

Teratogenic evaluation of Epichlorohydrin in the mouse and rat and glycidol in the mouse

Author:

Marks, T.A., Gerling, F.S. and Staples, R.E.

Year:

1982

Bibliographic source:

Journal of Toxicology and Environmental Health, 9:87-96, 1982

Materials and methods

Principles of method if other than guideline:

The present study was undertaken to determine whether epichlorohydrin and/or glycidol is teratogenic. Pregnant outbred albino rats (CD) and mice (CD-1) were given epichlorohydrin by gastric intubation on day 6-15 of gestation. The rats were killed on day 21 (day 18 for mice) and the offspring checked for gross, visceral, and skeletal malformations.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

epichlorohydrin

IUPAC Name:

epichlorohydrin

Constituent 2

Reference substance name:

1-chloro-2,3-epoxypropane

EC Number:

203-439-8

EC Name:

1-chloro-2,3-epoxypropane

Cas Number:

106-89-8

Molecular formula:

C3H5ClO

IUPAC Name:

2-(chloromethyl)oxirane

Details on test material:

Further read-across substance: glycidol (CAS 556-52-5)

Test animals

Species:

other: rat and mouse

Strain:

other: CD-rats and CD-1-mice

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- male and nulliparous female rats and mice
- Source: Charles River Breeding Laboratories (Wilmington, Mass.)
- Age at study initiation: 60 -90 d (mice)
- Weight at study initiation: 176-200 g (rats)
- Housing: Females werer housed in groups of 10 mice or 4 rats per polypropylene cage. The males were housed individually in polypropylene cages in seperated rooms by species.
- Diet (e.g. ad libitum): The animals were fed Purina Lab Chow ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature: 72-75 °F
- Humidity (%): 45-50 % relative humidity
- Photoperiod (hrs dark / hrs light): 12-h daily light cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

cotton seed oil

Details on exposure:

On day 6-15 of gestation, epichlorohydrin or glycidol was administered by gastric intubation at 40, 80, and 160 mg/kg/d in the rat study and at 80, 120, and 160 mg/kg/d in the mouse study. Glycidol was administered to mice at 100, 150, or 200 mg/kg/d. Each epichlorohydrin dosage except 120 mg/kg/d was studied independently of the other dosages in four or five replicates; 120 mg/kg/d was studied in only one replicate. The glycidol study was carried out in five replicates. Since no significant differences between replicates occurred, the results were combined in one table for each study. The dose that produced approximately 10% mortality was used as the maximum dose for each study; half this dose was used as a second dose. Later replicates included a third dose halfway between the high and low doses.
Epichlorohydrin was dissolved in cottonseed oil and glycidol was dissolved in sterile, distilled water (Gibco, Grand Island, N.Y.). In the rat study, the vehicle containing the epichlorohydrin was administered on a 0.1% (v/w) body weight basis. Epichlorohydrin and glycidol were administered to mice on a 1% (v/w) body weight basis. Since the animals and test (or control) groups were individually identified only by number, laboratory personnel knew neither the test agent nor the test group.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

After one week of acclimatisation, the mating period was begun by placing two females into each male's cage. The following morning, mice with vaginal plugs and rats with spermatozoa in their vaginal lavage (day 1 of gestation) were caged with the other animals found pregnant on that day.
Before d 1 of treatment, the dams were divided into experimental and control groups such that body weight differences between groups were minimized. The animals were individually identified by crystal violet markings and a number.

On day 6 -15 the animals were dosed with epichlorohydrin or glycidol.

Duration of treatment / exposure:

gestation day 6 - 15

Frequency of treatment:

once daily

Duration of test:

gestation day 6 - 15

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
40 mg/kg/day
Basis:
nominal conc.
Epichlorohydrin, rat study

Doses / concentrations 2

Remarks:

Doses / Concentrations:
80 mg/kg/day
Basis:
nominal conc.
Epichlorohydrin, rat and mouse study

Doses / concentrations 3

Remarks:

Doses / Concentrations:
120 mg/kg/day
Basis:
nominal conc.
Epichlorohydrin, mouse study

Doses / concentrations 4

Remarks:

Doses / Concentrations:
160 mg/kg/day
Basis:
nominal conc.
Epichlorohydrin, rat and mouse study

Doses / concentrations 5

Remarks:

Doses / Concentrations:
100 or 150 or 200 mg/kg/day
Basis:
nominal conc.
Glycidol, mouse study

Examinations

Maternal examinations:

On day 18 (d 21 for rats) of gestation, the mice were killed by cervical dislocation (the rats were first rendered unconscious with C02) and their reproductive status was determined.

Ovaries and uterine content:

Implantation sites in each uterine horn were counted and the general condition of each conceptus was recorded.

Fetal examinations:

Live fetuses were weighed individually, sexed internally, and examined for external anomalies (variations and malformations). Live fetuses weighing <0.5 g ( < 1.0 g for rats) or weighing less than two-thirds the mean of their larger littermates were designated as "stunted." At least one-third of the fetuses of each litter, as well as all stunted fetuses and those having external malformations, were examined for visceral alterations. The bodies of all fetuses were then processed for skeletal examination. The heads of fetuses subjected to visceral examination (with the exception of any fetuses that had external head malformations) were cut off at the base and prepared for freehand sectioning.
The average percent of malformed fetuses was calculated for each test group and for the vehicle controls as 100 * Sum of ((no of malformed fetuses in litter)/(no. of fetuses in litter))/(total no. of litters)

Statistics:

Statistically significant differences between groups were determined by the Mann-Whitney Latest or the Student t-test. Jonckheere's test was employed to determine the significance of dose-response relations. Two-tailed tests were performed and p< 0.05 was selected as the level of significance.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
In the rat study, epichlorohydrin at 160 mg/kg/d was lethal to 3 of 27 dams and also significantly (two-sided p < 0.05 versus the vehicle control group) increased the average liver weight of the dams. At 80 mg/kg/d, epichlorohydrin caused a significant decrease in the average weight gain during pregnancy.

Effect levels (maternal animals)

1

Dose descriptor:

other: not specified

Basis for effect level:

other: effect type not specified

Remarks on result:

other: no further information available

2

Dose descriptor:

other: not specified

Basis for effect level:

other: effect type not specified

Remarks on result:

other: no further information available

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
At 80 mg/kg/d, no significant differences in the average percent of malformed fetuses occurred between the control (0.18%) and treated groups (<= 0.76 %). Only three malformations were observed in this study: hydronephrosis in a control fetus, agnathia in a fetus in the 40 mg/kg/d group, and general oedema in a fetus in the 160 mg/kg/d group.

Effect levels (fetuses)

Remarks on result:

other: not specified

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

In this study, the type and quantity of variations found were not out of the ordinary and therefore will not be presented or discussed in this report.

In the rat study, epichlorohydrin at 160 mg/kg/d was lethal to 3 of 27 dams and also significantly (two-sided p < 0.05 versus the vehicle control group) increased the average liver weight of the dams (Table 1). At 80 mg/kg/d, epichlorohydrin caused a significant decrease in the average weight gain during pregnancy. In spite of these indications of maternal toxicity, no significant differences in the average percent of malformed fetuses occurred between the control (0.18%) and treated groups (<= 0.76 %). Only three malformations were observed in this study: hydronephrosis in a control fetus, agnathia in a fetus in the 40 mg/kg*d group, and general edema in a fetus in the 160 mg/kg'd group.

TABLE 1. Effect of Epichlorohydrin on Reproduction in Rats^a
Measurement	Dose (mg/kg/d)^b
	Control	40	80	160
Number of dams receiving test agent^d	35	14	25	27
Number of dams alive on d 18	35	14	24	24
Number of dams pregnant^e	32	10	23	22
Average weight gain (g) during pregnancy^	95.0 ± 1.97	85.6 ± 4.48^g	84.0 ± 4.35^h	88.1 ± 2.84
Average liver weight (± SE)^e	15.0 + 0.35	14.9 ± 0.59	16.1 ± 0.33	16.4 ± 0.37^h
Number of implants (avg.)^j	436 (13.6)	135 (13.5)	316 (13.7)	288 (13.1)
Number of females with resorptions	16	4	11	12
Number of resorptions	24	4	17	16
Average percent resorptions per litter	5.5	3.0	5.4	5.6
Number of fetal deaths	0	0	0	0
Average percent fetal deaths per litter	0	0	0	0
Male/female live fetuses	230/182	66/65	166/132^j	128/144
Number of stunted fetuses	1	0	0	0
Average number of live fetuses per dam	12.9	13.1	13.0	12.3
Average fetal weight (g)^k	3.73 ± 0.051	3.82 ± 0.084	3.71 ± 0.080	3.70 ± 0.052
^aKiIled on d 21 of gestation after receiving epichlorohydrin (gavage) on d 6-15 of gestation.
^bFive of five dams died before d 4 of dosing at 240 mg/kg.
^cReceived vehicle (cottonseed oil) only.
^dThe criterion used to identify mated females was spermatozoa in the vaginal lavage.
^eIncludes dams with all resorptions.
^fDays 6-20 of gestation; excludes dams with all resorptions; mean ± SE.
^gFive dams were omitted because of failure to record their weights on d 6.
^hTwo-sidedp <0.05 versus vehicle control group.
^iPer pregnant rat.
^jThe sex of one fetus was not determined.
^kStunted but- not dead fetuses were included; average of mean fetus weights per litter ± SE.

In the mouse study, epichlorohydrin at 160 mg/kg/d was lethal to 3 of 32 dams Table 2). No significant adverse effects on the average weight gain during pregnancy occurred at any of the doses tested. The 120 mg/kg/d dose did produce a significant increase in the average liver weight of the dams. This dose, as well as 160 mg/kg/d, led to a significant decrease in average fetal weight. The effect of epichlorohydrin on this parameter, as well as on average dam liver weight, also showed a significant (p< 0.005) trend with increasing dose levels (Jonckheere's test). Nonetheless, this compound did not cause a significant increase in the average percent of malformed fetuses at any of the doses tested (<= 3.5 %) as compared with the vehicle control group (0.23%).

No malformations were observed in the group receiving 120 mg/kg/d. One fetus in the control group had gastroschisis, ectocardia, and no right carotid artery. Three fetuses in the 80 mg/kg/d group had exencephaly with one or both eyes open, and one of these also had gastroschisis and cleft face. Another fetus in this group had fused arches, fused centra, and fused ribs. These four fetuses were in four litters.

In the 160 mg/kg/d group, five fetuses (four in the same litter) had exencephaly and bilateral open eyes; two fetuses (separate litters) had cleft palate; and another fetus had one eye open. A total of eight malformed fetuses in four litters was observed at this dose.

TABLE 2.Effect of Epichlorohydrin on Reproduction in Mice^a
Measurement	Dose (mg/kg/d)
	Control^b	80	120	160
Number of dams receiving test agent	49	31	24	32
Number of dams alive on d 18	49	31	24	29
Number of dams pregnant^c	40	25	22^d	20
Average weight gain (g) during pregnancy^e	17.5 ± 0.47	19.9 ± 0.79^f	19.1 ± 0.84^f	15.2 ± 1.44
Average liver weight (±SE)^c,g	2.37 ± 0.32^h	2.47 ± 0.054	2.52 ± 0.089^f	2.50 ± 0.119
Total number of implants	509	325	288	239
Average number of implants^i	12.7	13.0	13.1	12.0
Number of resporptions	46	23.0	44	22
Percent resorptions of total number of implants	9.0	7.1	15.3	9.2
Number of fetal deaths	8	1	6	8
Percent fetal deaths of total number of implants	1.6	0.31	2.1	3.1
Male/female live fetuses	212/243	169/132	128/110	108/101
Number of stunted fetuses	2	4	0	6^j
Average number of live fetuses per dam^e	11.4	12.0	11.9	10.5
Average fetal weight (g)^g,k	0.991 ± 0.011	0.990 ± 0.021	0.925 ± 0.022^f	0.898 ± 0.034^f
^aKilled on d 18 of gestation after receiving epichlororhydrin (gavage) on d 6-15 of gestation.
^bReceived vehicle (cottonseed oil) only.
^cIncludes dams with all resorptions.
^dTwo dams had all resorptions (implant sites).
^eDays 6-15 of gestation; excludes dams with all resorptions; mean ± SE.
^fTwo-sidedp <0.05 versus vehicle control group.
^gSignificant trend (two-sidedp <0.005 versus vehicle control group) as dose increased (Jonckheere's test).
^hOne liver weight not determined.
^iPer pregnant mouse.
^jFive of these fetuses were in one litter.
^kStunted but not dead fetuses were included; average mean fetus weights per litter ± SE.

Glycidol produced a significant number of stunted mouse fetuses in the group receiving 200 mg/kg/d (Table 3), but all of the stunted fetuses were in the same litter. In addition, 5 of the 30 females given this dose died, or were moribund and had to be sacrificed by d 16 of gestation. Two other dams experienced ataxia on one or more days during the treatment period.

Although a statistically significant increase in the average percent of malformed fetuses was not obtained as the result of glycidol treatment, 6 of the 15 stunted littermates in the 200 mg/kg/d groups had cleft palate. Since these cleft palates were found in stunted fetuses, they were not scored as malformations. Thus, even at 200 mg/kg/d, a dose that killed 5 of the 30 dams treated, glycidol did not cause a significant increase in the average percent of malformed fetuses (<= 0.66 %) as compared with the vehicle control group (0.23%). In this study, one fetus in the control group had fused sternebrae; one fetus in the 100 mg/kg/d dose group had meningocele and a small hole in the heart, and another in a second litter had fused sternebrae; and one fetus in the 200 mg/kg/d group had cleft palate. Glycidol at 500 mg/kg/d was either lethal or resulted in morbidity in 9 of 9 pregnant mice by day 10 of gestation.

TABLE 3. Effect of Glycidol on Reproduction in Mice^a
Measurement	Dose (mg/kg/d)^b
	Control	100	150	200
Number of dams receiving test agent^d	32	37	31	30
Number of dams alive on d 18	32	37	31	25
Number of dams pregnant	29	34	30	21
Average weight gain (g) during pregnancy^e	17.1 ± 0.832	19.3 ± 0.522	19.8 ± 0.600	17.0 ± 1.25
Number of implants (avg.)^f	373 (12.9)	458 (13.5)	390 (13.0)	291 (13.9)
Number of females with resorptions	23	28	21	17
Number of resorptions	41	50	32	34
Average percent resorptions per litter	11.0	10.9	8.2	11.7
Number of fetal deaths	3	10	5	3
Average percent fetal deaths per litter	0.8	2.2	1.3	1.0
Male/female live fetuses	187/142	190/208	183/170	114/140
Number of stunted fetuses	1	1	1	15^g,h
Average number of live fetuses per dam	11.3	11.7	11.8	12.1
Average fetal weight (g)^i	1.01 ± 0.015	0.984 ± 0.017	0.980 ± 0.016	0.955 ± 0.027
^aKilled on d 18 of gestation, after receiving glycidol (gavage) on d 6-15 of gestation.
^bNine of nine dams receiving glycidol at 400 mg/kg died or were sacrificed (near death) by d 10.
^cReceived vehicle (sterile distilled water) only.
^dVaginal plug was the criterion used to identify mated females.
^eDays 6-20 of gestation; mean ± SE.
^fPer pregnant mouse.
^gAIl fetuses were in the same litter.
^hTwo-sidedp <0.05 versus vehicle control.
^iStunted but not dead fetuses were included; average of mean fetus weights per litter ± SE.

Applicant's summary and conclusion

Conclusions:

The results of the present study indicate that epichlorohydrin is not teratogenic in albino mice and rats. The 160 mg/kg/d dose was lethal to some of the pregnant rats. The rat dams experienced significant reductions in weight gain with doses as low as 80 mg/kg/d. However, the average foetal weight, average number of live foetuses per day, average per cent of resorptions per litter, and number of stunted fetuses were not significantly affected by this compound. Thus epichlorohydrin apparently was not toxic to the rat embryo or foetus, even at doses that were toxic to the pregnant rat. Epichlorohydrin did cause a statistically significant decrease in average foetal (mouse) weight at 120 and 160 mg/kg/d. In addition, the overall dose-response effect on this parameter was also statistically significant (Jonckheere's test). The effects were confounded by the fact that both dose levels caused maternal toxicity. The 160 mg/kg/d dose killed 3 of 32 dams and the 120 mg/kg/d dose produced a statistically significant increase in the average liver weight in the treated mice. Thus signs of embryotoxicity were observed at doses that were toxic to the dam.

Executive summary:

Pregnant outbred albino rats (CD) and mice (CD-1) were given epichlorohydrin by gastric intubation on day 6-15 of gestation (Marks, 1982). The rats were killed on day 21 (day 18 for mice) and the offspring checked for gross, visceral, and skeletal malformations. Epichlorohydrin caused a significant reduction in the weight gain of pregnant rats at 80 mg/kg/d as compared with the control group treated only with the vehicle. However, there was no evidence of teratogenicity in the rat fetuses even at a dose level (160 mg/kg/d) that caused the death of some of the treated dams. Epichlorohydrin also did not produce a statistically significant increase in the average percent of malformed mouse foetuses, even at 160 mg/kg/d, a dose that killed 3 of 32 treated dams. The 120 and 160 mg/kg/d levels did cause a significant (p < 0.05) reduction in the average foetal weight as compared with controls. In addition, the 120 mg/kg/d dose produced a statistically significant increase in the liver weight of the pregnant mouse. These observations indicate that the 120 and 160 mg/kg/d dose levels were toxic toward the dams and their unborn offspring. In a similar mouse study, glycidol showed no evidence of teratogenicity. There was a significant increase in the number of stunted foetuses at 200 mg/kg/d, but all of these were present in a single litter. Further, the same dose killed 5 of 30 dams.