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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

supporting study

Study period:

20 July - 18 October 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP/Guideline study

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:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5450 (Rodent Dominant Lethal Assay)

GLP compliance:

yes

Type of assay:

rodent dominant lethal assay

Test material

Test material information

Constituent 1

Reference substance name:

3,6,9-trioxaundecane-1,11-diol

EC Number:

203-989-9

EC Name:

3,6,9-trioxaundecane-1,11-diol

Cas Number:

112-60-7

Molecular formula:

C8H18O5

IUPAC Name:

2,2'-[oxybis(ethane-2,1-diyloxy)]diethanol

Test material form:

not specified

Details on test material:

Tetraethylene glycol (Lot Number 118-175121) was obtained from Union Carbide Corporation, Texas City, Texas. The purity of the test substance as indicated by the supplier was 99.89%. Analyses conducted by the Union Carbide Chemicals and Plastics Technical Center, South Charleston, WV, on samples of the test substance, obtained both before and after the treatment period, confirmed that the test substance was approximately 99.7% pure and that no significant compositional changes occurred while the study progressed. Gas chromatography-mass spectrometry (GC/MS) and nuclear magnetic resonance spectroscopy (NMR) techniques were independently used to confirm its identity.

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

One hundred-twenty six (126) virgin male Fischer 344 [CDF@(F344)/CrlBR] inbred albino rats, were obtained from Charles River Breeding Laboratories, Inc. (Kingston, NY) in one shipment on July 2, 1990; they were 70 days old upon arrival (birth date April 23, 1990). Ten shipments of approximately 230 virgin female rats each of the same strain were also received from the same supplier; the first shipment arrived at the same time as the males; subsequent shipments arrived at approximately weekly intervals. Shipment one contained 231 females and arrived on July 2, 1990 (age 56-63 days upon arrival, date of birth May 3, 1990). Shipment two arrived on July 23, 1990, containing 230 females (age 63 days old upon arrival, date of birth May 21, 1990). Shipment three arrived on August 1, 1990, containing 228 females (age 70-75 days old, date of birth May 20, 1990). Shipments four and five were combined to arrive on the same date to allow for quality control to be performed on animals from shipment five. The combined shipments four and five arrived on August 6, 1990 k*"2 and contained 228 females (age 63 days old, birth date June 4, 1990) and 233 females (age 56-63 days old, birth date June 7, 1990), respectively. Shipment six arrived on August 20, 1990, containing 231 females (age 63 days old, birth date June 18, 1990). Shipment seven arrived on August 27, 1990 containing 230 females (age 63 days old, birth date June 25, 1990). Shipment eight also arrived on August 27, 1990, containing 230 females (age 56-63 days old, birth date June 28, 1990). Shipment nine arrived on September 12, 1990 and contained 80 females (approximate age 65 days; birth date July 10, 1990) and 120 females (69 days old; birth date July 6, 1990). Shipment ten was received on September 17, 1990 and contained 228 females (approximate age 63 days and birth date July 16, 1990). Since the females from shipments one, five and eight were held for approximately two (2) weeks before mating, they were slightly younger and lighter in weight, approximately 130-145 g upon arrival, than subsequent shipments. The females from the remaining shipments were approximately 145-160 g upon arrival. The Fischer 344 rat was selected by the Sponsor since it was employed in other studies with this test substance.

The males were received approximately two weeks before the initiation of the treatment period; shipment one of the females was received approximately three weeks prior to mating, and shipments five and eight of the females were received two weeks prior to mating. All other shipments of females were received approximately one week prior to their respective mating periods. During the pre-mating acclimation period, the animals were observed regularly for general health status and ability to adapt to the watering system. Body weights were measured at least once during the weeks prior to the mating period. Only male rats in good health whose weights were at least 200 g were used for the study. With the exception of shipment 9, only female rats in good health whose e! weights were at least 145 g were used for study; the minimum weight for shipment 9 females was 136 g.

Quality control was performed upon the receipt of the males, and females from shipments one and five. A quality control evaluation was performed on five animals/sex for serology and for fecal evaluations and on three animals/sex for histopathology. The quality control evaluation of these animals indicated that they were in good health and suitable for use in this study.

During the acclimation period and prior to mating, animals were housed separated by sex, one or two per cage, in stainless steel wire-mesh cages 30.5 x 15.5 x 18.0 cm (males) or 22.5 x 15.5 x 18.0 cm (females) mounted in steel racks in animal room 103. During Days 1-4 of the treatment period, the males were housed singly in stainless steel wire-mesh cages. On Day 4-5 of treatment, individual males were transferred to metabolism cages for collection of urine. After the treatment period, the males were housed in stainless steel wire-mesh cages (30.5 x 31.0 x 18.0 cm) with two females weekly, for mating. After successful mating, the females were housed one per cage in stainless steel wire-mesh cages mounted in stainless steel racks. Deotized* paperboard (Shepherd Specialty Papers, Inc., Kalamazoo, MI) was placed under each row of cages to collect solid and liquid
excrements. With the exception of the five days of dosing, an automatic watering system with demand control valves mounted on each rack provided water for study animals. Food was available ad libitum at all times. The light cycle in the animal rooms was 12 hours light/l2 hours dark. Each rat was assigned a unique number and was identified with a Monel* metal ear tag (Gey Band and Tag Co., Norristown, PA). Temperature and relative humidity were recorded continuously in the animal room. Room temperature was maintained at 68-75O~; relative humidity was maintained at 40-70% with the exception of two excursions totaling two and one-half hours during which the humidity ranged from 70-74%.

The water available to the animals during non-treatment periods was tap water (Municipal Authority of Westmoreland county, Greensburg, PA). The incoming water is analyzed approximately every 6 months for contaminants by the NUS Corporation (Pittsburgh, PA). The water quality analyses performed per EPA specifications include the 129 "priority" pollutants, which were identified in the Federal Register, Vol. 45, No. 98 Appendix D, Part 122. The food available to the animals was Purina Certified Ground Rodent Chow* #5002 (Batch numbers in the study notebook, Ralston Purina Co., Richmond, IN). The analyses of each feed batch for nutrient levels and for possible contaminants were performed by the supplier, Raltech Scientific Services, St. Louis, MO.

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

Drinking water solutions containing 5000, 25000 and 50000 ppm TTEG were prepared twice by adding the appropriate amount (grams) of processed tap water (CAS No. 7732-18-5) to the appropriate amount of TTEG placed in Nalgene" carboy, and mixing manually by swirling. Amber glass bottles with Nalgene stoppers were filled from the carboys and placed on the animal's caging.

Mean analytical concentrations of tetraethylene glycol in water for the five (5) days of exposure of the study males were 100.1 to 106.6 percent of nominal for the 5000, 25000 and 50000 ppm target concentrations. No test chemical was detected in the vehicle control (0 ppm) water with the minimum detection limit of approximately 500 ppm.

Homogeneity and stability studies performed on the 5000, 25000 and 50000 ppm dosing solutions indicated that the TTEG was uniformly distributed throughout the prepared solutions, and that solutions remained stable for at least 7 days when stored at room temperature.

Details on exposure:

One-hundred (100) virgin male Fischer 344 rats were assigned to three (3) treatment groups, one (1) positive control group and one (1) vehicle control group using a randomization procedure stratified by body weight, such that all five (5) groups did not differ substantially in mean body weight or body weight range. There were twenty (20) males per group. Two hundred (200) virgin female Fischer 344 rats from each shipment were similarly assigned to the five (5) groups by stratified randomization. The shipments of females were staggered (approximately one per week for ten weeks) to ensure that each set of females were approximately equivalent in age and weight when bred to the treated males.

Study males assigned to the test substance treatment group were administered TTEG in the drinking water for five (5) consecutive days (July 19-24, 1990) at target concentrations of 0, 5000, 25000 or 50000 ppm.

Males assigned to the positive control group received a single intraperitoneal injection of 0.5 mg/kg triethylenemelamine (TEM; CAS No. 51-18-3) 24 hours prior to the first weekly mating (corresponding to Day 5 of TTEG administration).

All males assigned to the vehicle control and TTEG treatment groups were weighed immediately prior to the first exposure, immediately after the last
exposure, and then weekly as any remaining females were removed from the cage and the two new females were added. Water consumption was measured during the five dosing days for control and test substance-treated groups and dosage (mg test substance/kg body weight/day) was calculated. Urine from control and TTEG-treated males was collected during the last 24 hours of the treatment period, and then analyzed for total volume, specific gravity, osmolality, pH , protein, glucose, ketones, bilirubin, occult blood and urobilinogen.

Males assigned to the positive control group were weighed immediately prior to the first exposure of TTEG-treated males, prior to dosing, and then weekly as the remaining females were removed from the cage and two new females were introduced. All males were examined daily for any clinical signs of toxicity.

On the day following the last exposure of the study males receiving the test substance (and 24 hours after injection of the positive control males), two naive (untreated) females were added to each male's cage. The females were checked twice daily for dropped or vaginal copulation plugs, and examined, if necessary, once daily (a.m.) by vaginal smear for detection of vaginal sperm. The date each female exhibited evidence of copulation was designated as gestational day (gd) 0 and recorded. The female was then removed from the male's cage and housed singly until scheduled sacrifice on gd 15. The females remained in the male's cage until there was evidence of copulation or until seven (7) days elapsed, whichever came first. If there was no evidence of copulation, then the sixth day of cohabitation was designated gd 0. After completion of the first week of breeding, two new naive females were added to each male's cage and observed and measured as described above. Two new females were added to each male's cage weekly for a total of ten (10) weeks to encompass the entire spermatogenic cycle. The dates of pairing of females with study males were July 25, 1990 (shipment one), August 1, 1990 (shipment two), August 8, 1990 (shipment three), August 15, 1990 (shipment four), August 22, 1990 (shipment five), August 29, 1990 (shipment six), September 5, 1990 (shipment seven) and September 12, 1990 (shipment eight), September 19, 1990 (shipment nine), and September 26, 1990 (shipment ten). All study females were examined daily for any clinical signs

Duration of treatment / exposure:

5 days

Frequency of treatment:

ad libitum

Post exposure period:

Males were held for 10 weeks after the last exposure to test material.

Doses / concentrations

Remarks:

Doses / Concentrations:
5000, 25000 and 50000 ppm in drinking water
Basis:
nominal in water

No. of animals per sex per dose:

20 males per group.

Groups of 40 females were placed with each group of males on a weekly basis (2 females to 1 male)

Control animals:

yes, concurrent vehicle

Positive control(s):

Males assigned to the positive control group received a single intraperitoneal injection of 0.5 mg/kg triethylenemelamine (TEM; CAS No. 51-18-3) 24 hours prior to the first weekly mating (corresponding to Day 5 of TTEG administration).

Examinations

Tissues and cell types examined:

In males, testes were weighed at necropsy.

In females, The gravid uterus, ovaries (including corpora lutes), cervix, vagina, and abdominal and thoracic organs and cavities of each pregnant animal were examined grossly. The number of live implants and early and late resorptions were recorded.

Details of tissue and slide preparation:

All study females were sacrificed on day 15 of gestation by carbon dioxide asphyxiation. The maternal body cavities were opened by a mid-sagittal
thoracolaparotomy. The gravid uterus, ovaries (including corpora lutes), cervix, vagina, and abdominal and thoracic organs and cavities of each pregnant animal were examined grossly. The uteri and attached ovaries and oviducts were removed from the peritoneal cavity and ovarian corpora lutea of pregnancy were counted. The uteri were palpated and, when necessary, dissected longitudinally to determine the status of the implantation sites. The number of live implants and early and late resorptions were recorded. Uteri from females that appeared nongravid were placed in a 10% ammonium sulfide solution (Salewski, 1964) for confirmation of pregnancy status. The females and their uterine contents were then discarded. Necropsies of females occurred from August 10, 1990 through October 18, 1990.

After the last (tenth) breeding period was completed, all study males were weighed, sacrificed by exsanguination following methoxyflurane anesthesia and subjected to a full necropsy. The testes were removed, weighed (combined), and preserved in 10% neutral buffered formalin. After the above procedures were completed, the males were discarded. The necropsy of the males occurred on October 4, 1990.

Evaluation criteria:

No additional information available.

Statistics:

The unit of comparison was the male and the pregnant female (Weil, 1970). Results of the quantitative continuous variables (e.g. male body weights) were intercompared for the three TTEG-treated groups and vehicle control group by use of Levene's test for equal variances (Levene, 1960), analysis of variance (ANOVA), and t-tests. The t-tests were used when the F value from the ANOVA was significant. When Levene's test indicated homogeneous variances, and the ANOVA was significant, the pooled t-test was used. When Levene's test indicated heterogeneous variances, a11 groups were compared by an ANOVA for unequal variances (Brown and Forsythe, 1974) followed, when necessary, by the separate variance t-test. The positive control group was compared to the vehicle control group using the previously described set of statistics.

Nonparametric data were examined statistically using the Kruskal-Wallis test (Sokal and Rohlf, 1969) followed by the Mann-Whitney U test (Sokal and Rohlf, 1969) when appropriate. Frequency data were compared using the Fisher's Exact Test (Sokal and Rohlf, 1969). For all statistical tests, the probability value of 0.05 (two-tailed) was used as the critical level of significance.

Dominant lethal parameters examined included:
No. impregnated females/no. females paired (mating index, females);
No. impregnating males/no. males paired (mating index, males);
No. pregnant females/no. females impregnated (fertility index, females);
No. males producing pregnant females/no. males impregnating (fertility index, males);
No. corpora lutea/pregnant female;
No. total implantations/pregnant female;
Preimplantation loss/pregnant female (%);
No. dead implantations (early and late resorptions)/pregnant female;
Postimplantation loss/pregnant female (%);
Proportion of females with one or more dead implantations;
Proportion of females with two or more dead implantations;
Ratio of dead implantations/total implantations;

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Males
Treatment and Necropsy Phases
There were no treatment-related clinical signs of toxicity during or subsequent to the five-day treatment period.

Body weights immediately prior to the start of test substance administration (Day 0) were equivalent across all groups, with the mean weight ranging from 241.9-244.7 gm. After the five day treatment period, body weights of all males administered TTEG were equivalent. In addition, no treatment-related effects on body weight were observed subsequent to treatment.

Males assigned to the positive control group were administered 0.5 mg/kg TEM on Day 5. On Day 13, approximately one week after intraperitoneal injection of the positive control, body weights of TEM-treated males were significantly reduced. Body weights of TEM-treated males remained reduced for approximately two weeks following TEM administration. For all other mating weeks (through Day 76) the body weights of the TEM-treated group were equivalent to control values.

For the dosing interval, Days 0-5, the 50000 ppm group exhibited weight loss.

Mean weight gain for TEM-treated males was significantly reduced for the first mating week (following treatment on Day 5). Subsequently, for Days 13-20 and 20-27, the TEM-treated males exhibited compensatory weight gains.

Water consumption was increased at 50000 and 25000 ppm throughout the dosing period,

Animals administered the 50000 pprn dosing solution consumed 5699 +/- 1341.2 mg TTEG/kg/day. Males administered 25000 pprn consumed an average of 2441 +/- 328.3 mg TTEG/kg/day and those given 5000 pprn in the drinking water consumed 425 +/- 44.8 mg TTEG/kg/day.

The increased urine volume observed at 50000 and 25000 pprn is consistent with increased water consumption observed in these dose groups. In addition, urine osmolality and pH were reduced at 50000 ppm. Reduction in urine pH was also noted at 25000 and 5000 ppm.

At necropsy, no treatment-related lesions were observed. There were no effects of treatment on terminal body weight, or on testes weights.

Mating and Reproductive Phase
The first and second weeks of mating reflect changes in sperm which are considered by Bateman (1960) and Leblond and Clermont (1952) to be those sperm cells maturing or being stored in the epididymis. The third, fourth and fifth weeks are associated with the stages of spermatid differentiation within the testes, Mating Weeks 6, 7 and 8 are associated with spermatocyte (meiotic) stages, and Weeks 9 and 10 are associated with stages of differentiation of the spermatogonia.

Weeks 1 and 2 - Mature Sperm Stages
There was no effect of TTEG treatment on the number of males impregnating females, the number of fertile males or the number of pregnant and plug- or sperm-positive females for Week 1. Mating and fertility indices indicated no effect of TTEG treatment.

In the TEM-treated group, the number of pregnant females was reduced. In addition, the number of females with one or more and two or more non-viable implants was increased. Calculated mating and fertility indices did not indicate any substantial effect of TEM treatment.

No apparent gestational effects of TTEG were noted. The dominant lethal fac tor (FL%) ranged from -8.9 to 10.1 in the control and TTEG-treated groups.

In the TEM-treated group, the numbers of total implants and viable implants was significantly reduced. Increased percentages of preimplantation loss, non-viable implants (based on significantly increased early resorptions), and postimplantation loss were observed. The ratio of dead implants to total implants was also increased in the TEM-treated group. The percentage of live fetuses was reduced and, consequently, the TEM-treated group exhibited significant dominant lethality (FL%=70.9) for the first mating week.

There were no effects of TTEG on reproductive parameters for Week 2 of mating.

For the TEM-treated group, the number of fertile males was reduced. In addition, females mated to TEM-treated males exhibited a reduced pregnancy rate, and the number of pregnant females with non-viable implants was increased. Calculated fertility indices were reduced in the TEM-treated
group.

There were no effects of TTEG on gestational parameters. There were no apparent dominant lethal effects of TTEG treatment. Statistical significance associated with the TTEG treated male data for Week 2 are considered spurious due to the lack of a dose response.

In the positive control group the number of corpora lutea are significantly reduced in females indicating a reduction in fertilization. The number of
total recognizable implantations was severely reduced in the TEM-treated group, such that no viable implants were observed--all implants were early or late resorptions. The calculated dominant lethal factor for implants resulting from the TEM-treated males was 100%.

Weeks 3, 4 and 5 - Spermatid Stages
There were no effects of TTEG treatment on reproductive parameters for Week 3.

TEM-treated males exhibited severely reduced fertility as indicated by the reduced number of pregnant females. Although the baseline number of at least one non-viable implant per female appears high (50%). However, all three females (100%) mated to TEM-treated males had non-viable implants, and two of the three pregnant females (66.7%) had two or more non-viable implants.

There was no effect of TTEG on gestational parameters for week 3. No dominant lethal effects were indicated in the TTEG-associated group.

The reduced number of corpora lutea observed in the TEM-treated group indicates reduced fertilization. The number of non-viable implantations from successful fertilizations was increased in the positive control group due to 100% early resorptions. No live fetuses were observed in the TEM-treated group, and the calculated FL% was consistent with this finding at 100%.

There were no effects on reproductive parameters in the TTEG groups for Week 3.

In the TEM-treated group, the number of fertile males remained reduced as reflected by a decreased number of pregnancies in mated females. In addition, the percentage of pregnant females with one or more and two or more non-viable implants was increased in the positive control group. Male and female fertility indices reflect the reduced number of pregnancies in the positive control group.

There were no effects observed in TTEG-treated groups for Week 4.

Data for TEM-associated females indicate reduced numbers of corpora lutea, total implants and viable implants. (Recall that for Weeks 2 and 3 no viable implants were observed.) The number of non-viable implants/early resorptions were increased in the positive control group as was the percentage preimplantation loss and the ratio of dead implants to total implants. The calculated dominant lethal factor, 87.8, indicates a significantly reduced number of live implantations.

No changes in reproductive parameters were observed in the TTEG-treated groups for Week 5.

In the TEM-treated group, the number of males impregnating females and the number of successful pregnancies were equivalent to control values. However, the number of females with one or more (or two or more) non-viable implants remained increased. Calculated mating and fertility indices for Week 5 were unaffected by TEM treatment.

There were no effects of TTEG treatment on gestational parameters for Week 5.

In females mated to TEM-treated males, there were no effects on the numbers of corpora lutea and total implants or on preimplantation loss. The number of viable implants was reduced and the number of non-viable implants was increased due primarily to an increase in early resorptions. The percentage of postimplantation loss, the ratio of dead implants to total implants and ultimately, the calculated dominant lethal factor, were increased in the positive control group.

Weeks 6, 7 and 8 - Spermatocyte Stages
Reproductive parameters for Week 6 are presented in Table 24. There were no effects of TTEG or TEM treatment on reproductive parameters for Week 6.

There were no treatment-related effects on gestational parameters including dominant lethality for Week 6.

For Week 7, there were no effects on reproductive parameters or on gestational parameters including dominant lethality in any dose groups including the TEM-treated group.

For Week 8, there were no effects of TTEG or TEM treatment on the number of males impregnating females or on the number of pregnancies. While the number of females with two or more non-viable implants appears increased in the 50000 ppm group, and in the positive control group, the percentages are within the ranges observed in the vehicle control group throughout the other study weeks. Mating and fertility indices are equivalent across all groups.

There were no dominant lethal effects in the TTEG treatment groups for Week 8.

In the TEM-associated group, there was a reduced number of viable implants, with the reduction due to slight reductions in corpora lutea and total number of implants, and slight increases in preimplantation loss and early resorptions. The slightly increased dominant lethal factor supports the
suggestion of effects on reproductive outcome in the positive control group for Week 8.

Weeks 9 and 10 - Differentiating Spermatogonia Stages
For Week 9, there were no effects of TTEG or TEM-treatment on reproductive parameters.

No statistical effects of treatment (TEM or TTEG] were observed for Week 9. However, there is a suggestion that percent postimplantation loss was slightly increased in the TEM-treated group.

For Week 10, there were no effects of TTEG or TEM treatment on reproductive parameters or on gestational parameters.

References:
Bateman, A. J. (1960). The induction of dominant lethal mutations in rats and mice with triethylenemelamine (TEM), Anat. Res., Camb. 1, 381-392.

Leblond, C. P. and Y. Clermont (1952). Spermiogenesis of Rat, Mouse, Hamster and Guinea Pig as Revealed by the "Periodic Acid-Fuchsin Sulfurous Acid Technique. Amer. J. Anat. 90(2), 167-215.

Any other information on results incl. tables

No additional information available.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
In conclusion, treatment of males for five days with 5000, 25000 or 50000 ppm TTEG in the drinking water and subsequent mating to naive (untreated) females over ten weeks produced evidence of toxicity in males at 50000 ppm and urinary findings consistent with an osmotic diuresis at 25000 and 50000 ppm, but no indications of subsequent reproductive or gestational effects. The "no observable effect level” (NOEL) for general toxicity was 25000 ppm. Under conditions of this study, TTEG did not exert a dominant lethal effect.

Executive summary:

Male Fischer 344 rats, 20 per group, were administered tetraethylene glycol (TTEG; CAS No. 112-60-7) in drinking water at

concentrations of 0 (water alone), 5000, 25000 or 50000 ppm for five (5) consecutive days. The respective average daily consumptions of TTEG were 425 ± 45, 2441 ± 328, and 5699 ± 1341 mg/kg. Selection of dose levels was based on a 5-day pilot study of TTEG administered in the drinking water to Fischer 344 rats (BRRC Project No. 90-22-74001). Twenty-four hours after the TTEG-treated water was replaced by tap water alone, males were bred to naive (unexposed) females of the same strain, one ma1e: two females. Each female was removed when evidence of copulation (copulation plug or vaginal smear) was observed or at the end of the mating week, whichever came first. Females were replaced weekly by a new pair of naive females for each male, for a total of ten consecutive weeks (i.e., 10 matings). The males were observed daily for clinical signs of toxicity, weighed daily throughout the treatment period and weekly during the mating period, and necropsied after the tenth week of mating. Urinalyses were conducted on urine collected on the fifth day of TTEG treatment. All females were observed daily during mating for evidence of copulation. From gestational day (gd) 0 (date of plug or sperm) to sacrifice on gd 15, females were examined for clinical signs. At sacrifice, ovarian corpora lutea and uterine implantation sites [non-live (early and late resorptions) and live implants] were counted and recorded. A concurrent positive-control group was also included in the study in order to validate the experimental design. The positive control group consisted of 20 male Fischer 344 rats which received a single intraperitoneal injection of 0.5 mg/kg triethylenemelamine (TEM) 24 hours prior to commencement of the ten-week mating regimen.

Males receiving 50000 ppm TTEG exhibited slightly reduced body weight gains after the five day treatment period. Body weights and weight gains were comparable to control values after the first mating week. Water consumption was increased in males at 25000 and 50000 ppm during the 5-day treatment period. Urinalyses indicated increased urine volume and decreased osmolality and pH at 50000 ppm, Urine volume was also increased at 25000 ppm, and decreased pH was noted at 25000 and 5000 ppm. There were no effects of treatment on gross observations at the time of necropsy of the males, nor were there any effects on terminal body weight or testes weights. Reproductive parameters, including the number of fertile males and the number of gravid females with viable implants, were unaffected by TTEG treatment. For the entire ten-week breeding regimen, no significant preimplantation loss or dominant lethal effects were observed in females mated to TTEG treated males.

Concurrent breeding of TEM-treated males indicated increased percentages of preimplantation and postimplantation loss, increased numbers of early resorptions, and significant dominant lethal effects for mating weeks corresponding to sperm stages of spermatogenesis (Weeks 1 and 2). For Mating Weeks 3 and 4 (which correspond to effects on spermatid stages of spermatogenesis), the rate of conception was reduced by 92.5% and 65%, respectively. For Week 3, the few successful fertilizations were non-viable and dominant lethality reached 100%. For Week 4, the greatly reduced rate of conception yielded only 12% viable fetuses (dominant lethal factor of 87.8). For early spermatid stages (Week 5) the number of conceptions reached control values but postimplantation loss and dominant lethality remained increased. TEM had no clearly measurable effect on meiotic stages of spermatogenesis (Weeks 6, 7 and 8), but slight postimplantation loss was suggestive for Week 9 (spermatogonial stages), There was no suggestion of reduced fertility or increased dominant lethality at the completion of the mating regimen (Week10).

In conclusion, treatment of males for five days with 5000, 25000 or 50000 ppm TTEG in the drinking water and subsequent

mating to naive (untreated) females over ten weeks produced evidence of toxicity in males at 50000 ppm and urinary findings consistent with an osmotic diuresis at 25000 and 50000 ppm, but no indications of subsequent reproductive or gestational effects. The "no observable effect level” (NOEL) for general toxicity was 25000 ppm. Under conditions of this study, TTEG did not exert a dominant lethal effect.