Reaction mass of 3,6,9-trioxaundecane-1,11-diol and 2,2'-oxydiethanol and 2,2'-(ethylenedioxy)diethanol and 3,6,9,12-tetraoxatetrade

Specific investigations: other studies

Administrative data

Endpoint:

specific investigations: other studies

Remarks:

Donor Recipient Carcinogenicity Study

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: sufficient documentation for interpretation of results

Justification for type of information:

Read across is based on the category approach. Please refer to attached category document.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1965

Report date:

1965

Materials and methods

Principles of method if other than guideline:

Donor recipient study to determine whether bladder tumors could develop as a result of the presence of stones or glass beads without the feeding of diethylene glycol. Therefore, DEG was included only in the diet of donor male rats to produce urinary calculi; then representative stones were implanted into recipient male or female rats.

GLP compliance:

no

Endpoint addressed:

carcinogenicity

Test material

Specific details on test material used for the study:

The diethylene glycol used in all phases of this study was obtained in 1959 from Union Carbide Corp. It was representative of commercial grade material and was neither purified nor redistilled.

Test animals

Species:

rat

Strain:

other: Carworth Farms Nelson (CFN)

Sex:

male/female

Administration / exposure

Control animals:

yes

Details on study design:

Donor groups: 1) 58 weanling DRN male rats that received 4% DEG; 2) 59 weanling Carworth Farms-Elias (CFE) male rats that received 4% DEG for 5 months and then 6% thereafter; 3) 19 weanling DFE male rats that received 4% DEG for 7 months and 6% thereafter; 4) 4% wealing rats from the feeding study who had stones at death

The first three donor groups were subjected to fluoroscopic examination and when stones were seen in the urinary bladder, they were surgically removed. After removal of the stones, the operated donors received control diet, containing no DEG.

An additional group of male and female CFE rats had their bladders surgically incised and sutured when they were 21 days of age; these rats were used to see if the performance of the operation per se would result in the earlier appearance of bladder stones and/or tumors. Eleven days post-operatively they were randomly assigned to receive either a 4 to 6% DEG of a control diet. The rats were fluoroscoped at monthly intervals but not reoperated to remove stones that formed.

Recipient rats received the sham operation only, received bladder implantation of a stone taken either at death or by operation from a donor rat, or received a glass bead by bladder implantation. Sub-groups would included both sexes, 3 different stocks, ages from weanling to older than one year at implantation, stones or beads varying from 1 mm to 4 mm in diameter and implants of more than 1 stone or bead.

The recipient rats did not receive DEG in their diets either pre- or post-operatively.

Examinations

Examinations:

Stone analysis: Representative calculi from rats on the feeding study, from donor rats and from recipient rats were analyzed by X-ray diffraction or by volumetric
determination of oxalate. Calculi that formed around the glass beads or in the surgically opened and closed rats were similarly sampled. The crystalline foms were analyzed using the Debye-Scherrer technique while Oxalate Content was determined by titration with solutions of potassium permanganate . If, on bisection, calculi appeared to be composed of a core and one or more outer layers, these were separated and separately analyzed for oxalate content.

Urine volume and acidity: The volume was measured and acidity determined on the urine of CFE male rats of two ages. One group of weanling rats was surgically
opened and sutured, rested one week and then started on diets containing 4.0 or 0.6 diethylene glycol. A second group of rats, 45 days of age, were fed diets containing 6.0 or 0.0% diethylene glycol. The former group received their diets for 280 days before urinary volume and acidity was measured on 8 different days, while the latter had these determinations made 5 times begining two days after they were started on their diets.

Results and discussion

Details on results:

Any of the 3 methods of bladder irritation; stone implant, glass bead implant, or simply the sham operation, resulted in a similar production of tumors as well as a similar production of stones.

Chemical composition of the stones was not related to tumor incidence as tumors resulted in feeding study and donor rats that had calcium oxalate stones; in stone recipient; glass bead and sham operation rats with nonaxalate stones; as well as in one rat with only a glass bead present at death.

Any other information on results incl. tables

12/58, 40/44, and 12/14 rats in the 1st, 2nd, and 3rd donor groups had stones surgically extracted. 2, 32, and 12 of these animals recovered from these operations. Complete removal of stones was not verified in group 2 and 13 of these rats had stones at death; none of the 13 had tumors but 2 of the remaining group 2 rats had bladder tumors but no stones. They had had 13 and 17 stones at operation. One of these tumors was the only bladder carcinoma found in any part of the study (others were papillomas).

20 male and 10 female rats were sham operated and fed DEG continously until death. 18 rats were sham operated only. No stones or tumors were found in the females or controls. The stone incidence in rats with surgery preceding the frist dose was practically identical to that in the donor groups; operation did not alter the occurrence of stones or tumors.

Stones and tumors were found in sham operated and glass bead implanted rats. Stones were found in 5/16, 4/8, 14/50, and 5/27 animals in male sham operated, female sham operated, male glass bead recipient, and female glass bead recipient groups, respectively.

In the stone recipient animals, 4 of 37 successfully stone implanted males and 3 of 30 successfully stone implanted females developed bladder tumors by the time of their deaths, 263 to 733 days after implantation. All of these 7 had bladder stones present (6 of the 7 had mutliple stones although only one was implanted) accompanied by bladder papilloma, at death. This verified that feeding DEG is unncessary for the production of bladder tumors - bladder surgery alone or glass bead or stone implantation is sufficient.

Stones from the nonoperated donors averaged 86.2% calcium oxalate. The bladder stones from the post-extraction donors were much lower in oxalate content averaging 16.1%. The latter indicates that some of the stones found in the rats at death as much as 328 days post-operation contained calcium oxalate. This probably indicates that, in some cases, all of the oxalate stones were not extracted at operation and that a nonoxalate coat was layered onto them, as no DEG was fed to these rats after thier operation. In the glass bead recipients and sham oepration rats that received no DEG at any time, the oxalate content of the stones was always below 2% and generally below 1%. The stone recipient rats all had implants of stones from DEG fed donor or feeding study rats; therefore, their impanted stones were composed primarily of calcium oxalate. In recipient rats with single or multiple stones at fate, outer layers of the stones were generally low in oxalate with the inner cores high in oxalate. The whole stones, 14 in number, analyzed from rats with multiple stones were low in oxalate content (mean of 2%). These outer layers and other stones, in the cases of multiple stones, were therefore not axalate but similar in composition to those in the bead insertion or sham operation rats. It is obvious that chemical composition of the stones was not related to tumor incidence as tumors resulted in feeding study and donor rats that had calcium oxalate stones; in stone recipient; glass bead and sham operation rats with nonaxalate stones; as well as in one rat with only a glass bead present at death.

Urinary volume of 6% DEG rats (45 days old at first dose) was almost exactly twice that of their controls (15.8 v7.3 ml/day) at days 2-9 after first dose while urinary acidity was increased to a lesser extent (5.78 v 6.02) (both significantly effected).

Stone and Tumor Summary
		Total number of rats	Rats with		Percentage of
			Stones at fate	Tumors at fate	Rats with stones	Rats with tumors	Rats with stones and tumors
Male Rats
Fed 4% or adjusted 4% DEG	Feeding study	60	18	1	30.0	1.7	5.6
	Donor, 1^st	58	18	2	31.0	3.4	11.1
	Donor, 2^nd& 3^rd	78	58	2	74.4	2.6	3.4
	Above donors – non operated	72	17	2	23.6	2.8	11.8
	Above donors – stones extracted; no DEG after extraction & survived > 2 days	46	43	2	93.5	4.4	4.6
	Sham operation before DEG	20	15	2	75.0	10.0	13.1
No DEG Fed	Stone recipients	37^X	18^M	4^M-4	48.6^M	10.8	22.2^M
	Glass bead recipients	50^X	14^Y	0^Y-1^Z	28.0	2.0^Z	0.0^Y
	Sham operation	16	5	0	31.2	0.0	0.0
Female Rats
Fed 4% or adjusted 4% DEG	Feeding study	56	0	0	0.0	0.0	0.0
No DEG Fed	Stone recipients	30^X	8^M	3^M-2	26.7^M	10.0	25.0^M
	Glass bead recipients	27^X	5^Y	0	18.5^Y	0.0	0.0
	Sham operation	8	4	1	50.0	12.5	25.0

X-successful stone or bead implants

M-no. or % with multiple stones; all 4 male and 2 of 3 females with tumors had multiple stones

Y-bead plus stones

Z-tumor in rat with bead but no stones

Applicant's summary and conclusion

Conclusions:

Evidence indicates that in rats bladder tumors were probably the result of mechanical irritation.