Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

two-generation reproductive toxicity
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study, no restrictions, fully adequate for assessment.

Data source

Referenceopen allclose all

Reference Type:
study report
Report date:
Reference Type:
Two-generation Reproductive Toxicity Study of Inhaled tertiary Amyl Methyl Ether (TAME) Vapor in CD Rats
Tyl RW, Myers CB, Marr MC, Fail PA, Seely JC, Elswick B, James A and Welsch F
Bibliographic source:
J Appl Toxicol 23:397-410.

Materials and methods

Test guideline
according to guideline
other: U.S. EPA Toxic Substances Control Act (TSCA) testing guidelines (US EPA, 1985), US EPA Office of Prevention, Pesticides and Toxic Substances (OPPTS) draft guidelines (US EPA, 1996)
GLP compliance:
Limit test:

Test material

Constituent 1
Reference substance name:
EC Number:
EC Name:
Cas Number:
1,1-dimethylpropyl methyl ether
Details on test material:
- Name of test material (as cited in study report): TAME
- Physical state: clear liquid
- Analytical purity: 98.8-98.9%, by GC and NMR
- Purity test date: 01-05-96
- Lot/batch No.: 95-01

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River Breeding Laboratories
- Age at study initiation: 7 weeks
- Weight at study initiation: 175-225 gram
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

- Temperature : 65-75°F
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
Type of inhalation exposure (if applicable):
whole body
unchanged (no vehicle)
Details on exposure:
TAME exposure concentrations were generated by metering liquid TAME from a stainless steel reservoir into a J-tube generator with an FMI pump. The FMI pump was calibrated and set to the nominal flow rate expected for the target exposure concentration. Nitrogen flowed up through the J-tube at a flow rate of approximately 20 L/min. The TAME was pumped into the upper portion of the J-tube and allowed to flow downward through glass beads, counter-current to the nitrogen flow. A heating jacket warmed the J-tube to approximately 40 to 60 oC. The heat and the increased surface area from the glass beads facilitated the vaporization of the TAME. A sight gauge at the bottom of the J-tube verified total vaporization of the TAME. The vaporized TAME was carried in the nitrogen stream from the top of the J-tube into a 'T" in the stainless steel, HEPA-filtered air inlet to the inhalation chambers. The "T" was located approximately three feet upstream of the chamber. The TAME vapor flowed into the "T" and counter-current to the HEPA-filtered chamber air flow to facilitate mixing of the TAME vapor with dilution air. The total chamber air flow through each chamber was maintained at approximately 220-250 L/min.
Four H-1000 inhalation exposure chambers (Lab Products, Maywood, NJ) were used for these exposures. One H-1000 was used for each target exposure concentration (0, 250, 1500 or 3000 ppm). Each H-1000 was located inside one eight cubic meter Hinners-style stainless steel and class inhalation exposure chamber. The H-1000 is constructed of stainless steel and glass with silicon seals on the doors.
Details on mating procedure:
Animals were mated randomly within treatment groups to produce F1-generation. F1- litters were culled to 10 pups on post-natal day (PND) 4 and weaned on PND 28. 30 animals/sex/dose were selected as parents of the F2-generation. The selected F1-weanlings were exposed 10 weeks with acquisition of vaginal patency and the assessment of preputial separation in males and vaginal cytology evaluated during the last three weeks. Results from these measurements triggered the measurement of anogenital distance in F2 offspring at birth. F1 animals were mated in a two-week mating period following the procedure used with F0 animals. At weaning 30 animals/sex/dose were selected for post-wean retention with no exposure through acquisition of vaginal patency in females and preputial separation in males.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The concentration of TAME inside the exposure chamber was confirmed using IR analysis (MIRAN 1A).
Duration of treatment / exposure:
Premating exposure period:
males: 5 days/week for 10 weeks (6 hours/day, 5 days/week)
females: 5 days/week for 10 weeks (6 hours/day, 5 d/week)

Exposure period:
males: mating, postmating (30 days)
frequency: 7 days/week during mating, then returning to a 5 days/week schedule until necropsy.

females: mating through gestational day 19, lactation (postnatal day 5 through 28)
frequency of treatment: 6 hours/day, 7 days/week throughout mating, gestation and lactation. F0 females were necropsied on post-natal day (PND) 28.
Frequency of treatment:
See section on Duration of treatment / exposure
Doses / concentrationsopen allclose all
Doses / Concentrations:
250, 1500 or 3000 ppm
other: target concentrations
Doses / Concentrations:
245(7), 1493(13), 2992(46) ppm (SD between brackets)
analytical conc.
No. of animals per sex per dose:
Each exposure group had 30 animals/sex
Control animals:
Details on study design:
The target exposure concentrations were 250, 1500, and 3000 ppm. The rationale for choosing these exposure concentrations was as follows. A previous developmental toxicity study in pregnant rats based on a range-finding study in pregnant rats which employed exposure concentrations of 0, 250, 1500 and 3500 ppm resulted in demonstrable maternal toxicity and developmental toxicity at 1500 and 3500 ppm, and no maternal toxicity or developmental toxicity at 250 ppm. In addition, a preliminary study on weanling rats (to model the beginning of the F1 exposure period) exposed to 3500 ppm for four days six hours per day, resulted in severe and persistent toxicity. The highest exposure concentration level, 3000 ppm, was therefore chosen to induce overt maternal and offspring toxicity, but not excessive mortality.


Parental animals: Observations and examinations:
Body weights, feed consumption and clinical signs were recorded daily.
Oestrous cyclicity (parental animals):
An andrological assessment was conducted to all parental males.
Vaginal cytology was evaluated for F0 females daily during the last three weeks of the 10-week prebreed exposure period.
Vaginal cytology was also performed in F1 females.
Sperm parameters (parental animals):
An andrological assessment was conducted to all parental males.
Testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology
Litter observations:
Up to three F1 weanlings/sex/litter were chosen for necropsy.
Up to three F2 weanlings/sex/litter were selected for necropsy with selected organ weights.
Postmortem examinations (parental animals):
F0 males were submitted to necropsy and evaluation of reproductive and other selected organs (high and control animals) after the delivery period.
F0 females weresubmitted to necropsy and histopathology of reproductive and other selected organs of high and control dose animals.
Postmortem examinations (offspring):
Up to three F1 weanlings/sex/litter were chosen for necropsy.
Up to three F2 weanlings/sex/litter were selected for necropsy with selected organ weights.
Statistical analysis used either the male, the female, the pregnant female or the litter as the unit of comparison. Appropriate parametric- or non-parametric tests were applied to the data after initial analysis using Bartlett's test for homogeneity of variances.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
no effects observed

Details on results (P0)

Systemic toxicity in adult F0 and F1
Adult systemic toxicity was present in F0 and F1 animals at 1500 and 3000 ppm. Both sexes and generations displayed ataxia and persistently reduced body weights at 3000 ppm. Body weights were reduced during gestation in F0 (GD 0-7, ~6%) and F1 (GD 0-20, ~9%) and during lactation in F0 (PND 0 – 4, 5-9%) and F1 (PND 0-14, 7-10%) at 3000 ppm. Ataxia was present in the 1500 ppm F0 animals but there were no effects on body weights or food efficiency. F1-dams had a reduction in lactation period weight gain. In necropsy, the liver weights (absolute and relative) were increased in the F0 and F1 males and less in females. In F0 males, the relative to body weight increase was at 1500 ppm 29% and 3000 ppm 33% and F0 females at 3000 ppm 24%. In F1 males, the relative liver weight increase was 22% in the 1500 ppm and 3000 ppm dose group. In F1 females, the relative liver weight increase at 3,000 ppm was 14%. The relative kidney were significantly increased in the 1500 F0 males (21%) and 3000 ppm F0 males (26%) and females (9%), while male absolute kidney weight was significantly increased at all levels (8-19%) and in females only 9% at 3000 ppm. F1 males, but not females, had increased kidney weight at 1500 ppm (18%) and 3000 ppm (22%). Relative but not absolute spleen weights were significantly increased in F1 males at 1,500 and 3000 ppm. Relative and absolute adrenal weights were significantly increased at the 3000 ppm group in males and females.

Reproductive toxicity in F0 animals
In F0 and F1 males, minimal effect on gonads were noted at 3000 ppm, which was expressed as increased relative but not absolute testes weight, probably due to reduced terminal body weights in this group. The absolute prostate weight was significantly reduced at 3000 ppm for F1 males but not for F0 males. At 3000 ppm, F1, but not F0 males had reduced sperm concentrations of the epididymis. Both the 1500 ppm (3.13%) and 3000 ppm (5.49%) F0 males had an increase of abnormal sperm counts compared to the concurrent control group (0 ppm (2.26%, 250 ppm (2.40%)), but not when compared to the historical values (range: 2.0 ± 0.3% to 6.1 ± 3.3%). The F1 males also had an increasing trend of abnormal sperm counts, although none of the figures was statistically significantly over the concurrent control group (0 ppm: 2.5%, 250 ppm: 2.7%, 1500 ppm: 3.9%, 3000 ppm: 3.5%). The treatment had no effect on mating or survival indices, on absolute testes weight, on absolute or relative epididymides or seminal vesicles with coagulating gland, on relative prostate weight, sperm motility, or sperm production. There were no treatment-related findings in the histopathology.

In females, no treatment-related change was noted in the vaginal cyclicity, or oestrous cycle length. Gestational length was significantly longer in the F1 1500 ppm females but this was probably of no toxicological significance, because the effect was not seen in the F1-3000 ppm group or the F0 females. No significant changes were seen on the number implantation sites per litter, on prenatal mortality index or the number of live or dead pups on PND 0.

Effect levels (P0)

open allclose all
Dose descriptor:
parental toxicity
Effect level:
250 ppm
Basis for effect level:
other: Changes in liver adrenal and kidney weights and reduction in body weight, general signs of toxicity to central nervous system, such as ataxia
Dose descriptor:
effects on reproductive toxicity
Effect level:
3 000 ppm
Basis for effect level:
other: no toxicological relevant effects were observed
Remarks on result:
other: Generation not specified (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

Reproductive toxicity in F0 and F1
In the offspring, the acquisition of preputial separation was significantly delayed in the 1500 and 3000 ppm F1 males and F2 males at 3000 ppm. Acquisition of vaginal patency was significantly delayed at 3000 ppm in F1 females and at 250 and 3000 ppm in F2 females. These effects were considered as the landmarks, which triggered the measurement of anogenital distance (AGD) in the F2 animals. The AGD was significantly decreased in both sexes of the F2 3000 ppm rats. This was accompanied and probably caused by a significant reduction (~ 8%) in body weights/litter in the F2 pups on PND 0. Although the 3000 ppm F1 body weights per litter were significantly lowered on the post-natal days 4, 7, 14, 21 and 28, the survival indices were not affected in the F1 offspring throughout lactation. The F1 1500 ppm animals had reduced litter body weights on PND 4 and 7 (females) and 14, 12 and 28. The 250 ppm F1 pup body weights per litter were reduced on PND 14, 21 and 28 (28 days, males only). At 3000 ppm, F2 offspring had reduced survival indices on PND 4 and 21 and the pup body weights were significantly reduced during lactation on PND 0, 4, 7, 14, 21 and 28. In the 1500 ppm group significantly reduced pup weights were observed on PND 14 and 21. There were no effects on F2 pups at 250 ppm. The increased offspring mortalities at 1500 and 3000 ppm were thought to have been due to the “overt toxicity and the compromised status” of the dams. The observations included lethargic or hypothermic pups with little or no milk in stomach, tail chewed off, thin fur and haematomas at various locations.

Effect levels (F1)

Dose descriptor:
toxicity to offspring
Effect level:
250 ppm
Basis for effect level:
other: see 'Remark'

Results: F2 generation

Effect levels (F2)

Dose descriptor:
toxicity to offspring
Effect level:
250 ppm
Basis for effect level:
other: see effects on F1 generation

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Endocrine disruption potential

The authors state that TAME changes seen in the reproductive landmarks, the acquisition of preputial separation and vaginal patency cannot be interpreted as endocrine disruption activity. This is because if TAME would have androgenic or anti-androgenic or estrogenic or anti-estrogenic activity, depending on the activity and sex in question one of the landmarks would have been delayed while the other one was accelerated or not affected. For example, if TAME was an estrogenic antagonist, vaginal patency would be delayed and preputial separation unchanged. The same is true for the reductions seen in the anogenital distance. If TAME was, e.g., an anti-androgen, males only would be expected to exhibit shorter AGD, whereas females would be unaffected or have a longer AGD. Neither was true in this case. On the other hand, if the preputial separation and AGD were earlier dismissed as evidence for reproductive toxicity by the authors because the differences were said to have been caused by significantly different pup weights on PND 0, then their use as negative evidence for endocrine disruption is questionable.

Applicant's summary and conclusion