reaction mass of sodium acetate, monoethylene glycol and diethylene glycol

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

three-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Assessing the effect of EG on fertility and general reproductive performance in male and female rats.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Young adult nulliparous Fischer 344 rats were housed two per cage in stainless-steel wire cages at 20-24°C with controlled lighting (12 hr light) and fed Purina Formulab and city water ad libitum. During mating, each male was housed with 2 females. After mating and during lactation, the female rats were housed individiually in plastic showbox cages with hardwood chips for nesting. At study initiation the animals were randomly assigned to receive dietary EG at approximate dosages of 1.0, 0.2, or 0.04 g/kg/day.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

Fresh diet was prepared every 2 weeks with the percentage of EG adjusted, based on the group mean body weight and food consumption, so as to maintain a relatively constant dosage level. However, the concentration of EG in the diet was not changed during gestation or during the first week of lactation, but was reduced two- and threefold during the second and third weeks of lactation, respectively, to adjust for increased food consumption by the dams. This change in concentration as based on earlier unpublished results from the laboratory. Increased food consumption during lactation has since been reported in another study performed at the laboratory.

Details on mating procedure:

At approximately 100 days of age, 10 males were added to 20 females in each dosage group. The date of parturition and the number of live and dead newborn were recorded for each litter. The appearance and behavior of dams and pups were observed daily. Litter size was randomly reduced to 10,
if necessary, on Day 4 postpartum. Offspring were weighed as litters at 4 and 14 days and individualiy at 21 days postpartum, the day they were weaned.
F1 rats were randomly selected within each dosage group for the next mating. Each litter was represented except for those conceived very late in the mating period. The F1 and F2 rats were treated as described for the F0 animals until approximately 100 days of age, at which time the animals were cohabited. Brother and sister matings were avoided for each generation.

Duration of treatment / exposure:

3 generations

Frequency of treatment:

daily

Dose / conc.:

40 mg/kg diet

Dose / conc.:

200 mg/kg diet

Dose / conc.:

1 000 mg/kg diet

No. of animals per sex per dose:

30

Control animals:

yes, concurrent no treatment

Details on study design:

Reproductive function was tested in a protocol that allowed continuous breeding during a 14-week period. Small, but statistically significant, decreases were found in the numbers of litters per fertile pair and in live pups per litter in the 1.0% dose group and in live pup weight at the 0.5 and 1.0% concentrations. Facial anomalies were noted in the offspring of mice at the high concentration of EG. Skeletal examination revealed a pattern of reduction in the size of bones in the skull, fused ribs, and abnormally shaped sternebrae and vertebrae in the highest dose group.

Positive control:

yes

Postmortem examinations (parental animals):

Necropsies were performed on five males and five females randomly selected from each dosage level of the F2 parents and the F3 weanlings. Microscopic examulations were performed on sections of liver, kidneys, lung, heart, adrenals, thyroid, trachea, accessory sex glands, adipose tissue, lymph nodes, pituitary, thymus, and testes and epididymis, or uterus and ovaries.

Statistics:

Continuous data such as body weights were compared by analysis of variance validated by Bartlett's test for homogeneity of variance. Duncan's multiple range test wes used to identify individual mean differences when indicated by a significant F value. Where Bartlett's test indicated heterogeneous variances, t tests for equal or unequal variances were used to delineate differences between groups. Pup weights were compared by the metbod of Weil (Weil, 1970). Discontinuous data such as implantations and reproductive indices were compared by a multiple sum of ranks test. Frequency data were compared by the X2 test and by Fisher's exact test. The following reproductive indices were calculated and evaluated statistically by the previously described nonparametric methods: fertility index (male and female), days from first mating to parturition, gestation index (fraction of pregnancies that resulted in litters with live pups), gestation survival index (fraction of newborn pups alive at birth), 0 to 4-day survival index, 4 to 14-day survival index, 4 to 21day survival index. The last four indices are summarized in the tables as means for ease of understanding and presentation, although the nonparametric statistical methods did not include a comparison of means.

Reproductive indices:

yes

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

No treatment-related effect was observed for any of the reproductive indices.

The calculated dosages based on the nominal concentrations of EG in the diet were generally very close to the dosage goals. The weekly calculated dosages ranged from 1.0 to 1.3, 0.2 to 0.3, and 0.04 to 0.05 g/kg/day for males and from 0.9 to 1.2, 0.2 to 0.3, and 0.04 to 0.06 g/kg/day for females.

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

There were no reproductive effects associated with the inclusion of as much as 1.0 g/kg/day of EG in the diet.

Critical effects observed:

no

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathologic findings: There were no treatment-related histopathologic findings in F2 parents. Although the kidney has been shown to be the primary target organ for EG-induced toxicity, there was no increase in the incidence or severity of kidney lesions in this study. One high dose F2 animal of each sex had mild focal interstitial nephritis. However, this condition was also seen in a control male and a control female. Unilateral hydronephrosis occurred in another high-dose F2 male.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

EG treatment did not affect neonatal body weight at days 4, 14, or 21 post partum.

Dose descriptor:

NOAEL

Generation:

F1

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Description (incidence and severity):

There were no treatment-related histopathologic findings in F3 weanlings. Although the kidney has been shown to be the primary target organ for EG-induced toxicity, there was no increase in the incidence or severity of kidney lesions in this study. One high dose F2 animal of each sex had mild focal interstitial nephritis. However, this condition was also seen in a control male and a control female. IMild focal tubular hyperplasia was observed in one high-dose male F3 pup but was also diagnosed in two control male pups.

Dose descriptor:

NOAEL

Generation:

F2

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

no

Reproductive Indices

		1.0 g/kg/d	0.2 g/kg/d	0.04 g/kg/d	0.0A	0.0B
F0 -> F1	Fertility index (%)	100	90	100	90	90
	Male	95	90	90	75	90
	Female	100	100	100	100	100
F1 -> F2	Fertility index (%)	100	100	90	90	90
	Male	85	95	85	90	85
	Female	100	100	100	100	94
F2 -> F3	Fertility index (%)	100	90	100	80	80
	Male	90	75	85	80	70
	Female	100	100	100	100	100

Neonatal body weight at day 21

		1.0 g/kg/d	0.2 g/kg/d	0.04 g/kg/d	0.0A	0.0B
F1 pups	males	30.6 +/- 4.5	30.9 +/- 4.9	30.7 +/- 6.4	30.6 +/- 3.6	27.9 +/- 4.3
	females	29.0 +/- 4.5	29.2 +/- 4.5	29.5 +/- 4.7	27.9 +/- 3.3	27.0 +/- 3.5
F2 pups	males	32.8 +/- 3.5	30.9 +/- 5.8	29.3 +/- 4.7	30.0 +/- 4.0	28.8 +/- 4.3
	females	30.8 +/- 3.4	30.2 +/- 4.9	28.8 +/- 3.8	28.5 +/- 3.1	27.5 +/- 3.4
F3 pups	males	30.2 +/- 4.0	30.9 +/- 4.0	30.9 +/- 4.0	32.0 +/- 3.9	30.2 +/- 4.6
	females	28.6 +/- 3.8	28.2 +/- 3.4	29.7 +/- 4.0	30.1 +/- 3.5	27.7 +/- 3.9

Conclusions:

In conclusion, there were no reproductive effects associated with the inclusion of as much as 1.0 g/kg/day of MEG in the diet.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1986

Materials and methods

Principles of method if other than guideline:

Assessing the possible effects of ethylene glycol for reproduction and dominant lethal mutagenesis. Study performed as an add-on to a 3-generation reproductive toxicity test.

GLP compliance:

not specified

Type of assay:

rodent dominant lethal assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethane-1,2-diol

EC Number:

203-473-3

EC Name:

Ethane-1,2-diol

Cas Number:

107-21-1

Molecular formula:

C2H6O2

IUPAC Name:

1,2-ethanediol

Constituent 2

Reference substance name:

ethan-1,2-diol

IUPAC Name:

ethan-1,2-diol

Details on test material:

purity: 99.93%

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Further details see record in Reproduction Toxicity.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

Further details see record in Reproduction Toxicity.

Details on exposure:

Further details see record in Reproduction Toxicity.

Duration of treatment / exposure:

Further details see record in Reproduction Toxicity.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

15

Positive control(s):

Further details see record in Reproduction Toxicity.

Examinations

Tissues and cell types examined:

Further details see record in Reproduction Toxicity.

Details of tissue and slide preparation:

Further details see record in Reproduction Toxicity.

Statistics:

Continuous data such as body weights were compared by analysis of variance validated by Bartlett's test for homogeneity of variance. Duncan's multiple range test wes used to identify individual mean differences when indicated by a significant F value. Where Bartlett's test indicated heterogeneous variances, t tests for equal or unequal variances were used to delineate differences between groups. Pup weights were compared by the method of Weil (Weil, 1970). Discontinuous data such as implantations and reproductive indices were compared by a multiple sum of ranks test. Frequency data were compared by the X2 test and by Fisher's exact test. The following reproductive indices were calculated and evaluated statistically by the previously described nonparametric methods: fertility index (male and female), days from first mating to parturition, gestation index (fraction of pregnancies that resulted in litters with live pups), gestation survival index (fraction of newborn pups alive at birth), 0 to 4-day survival index, 4 to 14-day survival index, 4 to 21day survival index. The last four indices are summarized in the tables as means for ease of understanding and presentation, although the nonparametric statistical methods did not include a comparison of means.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Ethylene glycol administration did not lead to statistically significant adverse effects on any of the parameters measured in the three mating intervals. Slight apparent increases in the dominant lethal mutation index, observed during the week 2 mating for the high-dose (1.0 g/kg/day) group and during the week 3 mating for the low dose (0.04 g/kg/day) group, were probably random occurrences unrelated to EG treatment. This interpretation is consistent with the absence of a dose-response relationship and the fact that a negative index of similar magnitude (-8.2%) was observed for the low dose group in the week 2 mating. When compared to the combined control groups, significant decreases were observed as a result of TEM treatment for the number of females with implants, the total number of implants, and the number of live implants.

Any other information on results incl. tables

Summary of dominant lethal mutagenesis data:

Dose	Mating interval (week)	Females with implants		Total implants		Dead implants		Live implants		Dominant lethal mutations (%)
		N	%	N	%	N	%	N	%
1 g/kg/d	1	13	87.7	128	9.8	6	0.46	122	9.4	0
	2	13	86.7	107	8.2	10	0.77	97	7.5	11.0
	3	13	86.7	116	8.9	7	0.54	109	8.4	-1.2
0.2 g/kg/d	1	11	73.3	108	9.8	10	0.91	98	8.9	5.3
	2	14	93.3	132	9.4	14	1.00	118	8.4	1.2
	3	14	93.3	134	9.6	19	1.36	115	8.2	1.2
0.04 g/kg/d	1	11	73.3	107	9.7	8	0.73	99	9.0	4.2
	2	13	86.7	131	10.1	11	0.85	120	9.2	-8.2
	3	14	93.3	118	8.4	18	1.29	100	7.1	14.5
0.0A	1	13	86.7	116	8.9	10	0.77	106	8.1
	2	13	86.7	106	8.1	7	0.54	99	7.6
	3	13	86.7	123	9.5	12	0.92	111	8.5
0.0B	1	10	66.7	113	11.3	3	0.30	110	11.0
	2	11	73.3	109	9.9	5	0.45	104	9.4
	3	11	73.3	100	9.1	11	1.00	89	8.1
TEM (0.5 mg/kg)	1	3	20.0	7	2.3	3	1.00	4	0.75	92.0
	2	8	53.3	15	1.9	4	0.50	11	1.37	83.9
	3	1	6.7	1	1.0	1	1.00	0	0.00.	100.0

Applicant's summary and conclusion

Conclusions:

The absence of a genotoxic effect in the dominant lethal study is consistent with negative genotoxicity results from other studies with EG. Although mutagenic activity has been demonstrated in some studies the divergent results may be explained in part by the presence of impurities in the samples tested. Unpublished data from our laboratory indicate that small amounts of impurities such as oxidation products and storage contaminants such as iron compounds may substantially affect the results of in vitro mutagenicity assays of EG samples. Of course, different mutagenicity assays have distinct end points and varying sensitivities. Therefore, when several different tests are performed, contrasting results are not unexpected.