DL-hexane-1,2-diol

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: publication is on various substances, where tests were conducted with GLP.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

Reproductive Assessment by Continuous Breeding (RACB) study

Deviations:

not specified

Principles of method if other than guideline:

The mice were exposed to the chemical for a 7-day premating period, and were then randomly grouped as mating pairs and cohabited and treated continuously for 14-week. At the end of 14 weeks the male and female are separated and the last litter delivered is used for the second generation. F1 animals from the control group and treated animals exposed to the test chemical at the treatment levels indicated. The crossover mating was not done if significant reproductive effects were not observed in the continuous breeding phase.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Propane-1,2-diol

EC Number:

200-338-0

EC Name:

Propane-1,2-diol

Cas Number:

57-55-6

IUPAC Name:

propylene glycol

Test material form:

not specified

Details on test material:

Chemicals were evaluated for purity and for stability in the dosing vehicle for up to 14 days, Any impurity which equaled 1.6% or more of the product was identified. Most chemicals were administered in the diet or drinking water.

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

COBS Crl:CD-l (ICR)BR outbred Swiss albino mice (6 weeks of age) were used. Two males and two females were killed and their sera evaluated for antibodies against 11 mouse viruses. All sera were negative for viral antibodies. All study animals were individually identified and assigned to treatment groups using a stratified randomization procedure based on body weights.
Males and females were group housed by sex in solid- bottom polypropylene or polycarbonate cages with stainless-steel wire lids, during the quarantine and the 1- week premating periods. Subsequently, the animals were housed as breeding pairs or individually. Ad-Sorb-Dn bedding was used in all cages. Deionized/filtered water and ground rodent chow were provided ad libitum. Automatically controlled photoperiods were 14 hr light/10 hr dark (lights on from 0700 to 2100 hr), and temperature was maintained at 23 ± 2°C. Cages were sanitized weekly using detergent and 180 °F water.

Administration / exposure

Route of administration:

other: oral: feed or water

Vehicle:

unchanged (no vehicle)

Details on exposure:

The mice were exposed to the chemical for a 7-day premating period, and were then randomly grouped as mating pairs and cohabited and treated continuously for 98 days.

Details on mating procedure:

The mating trial begins within a week of the start of treatment and cohabitation continues for 14 weeks to maximize the data collected on fertility. The animals are housed as breeding pairs, one male and one female per cage, for the 14-week period and the offspring of as many as five litters are evaluated during the test. At the end of 14 weeks the male and female are separated and the last litter delivered is used for the second generation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each concentration of each compound was mixed separately at periodic intervals depending on the stability of that chemical. For dosed feed studies, a stock mixture of dosed feed was prepared by weighing an aliquot of test chemical and mixing it with, a small amount of feed in a beaker. Diets were prepared in NIH-07 diet6 or Purina certified chow. The stock mixture was then added to a prewedghed portion of untreated ground feed and mixed in a Patterson-Kelly 8-quart stainless-steel V-type blender and blended for at least 20 min. Diets were stored at -20 or 4 °C until used. For each chemical, an aliquot of each formulation, the feed control, and the bulk material were sent to an independent laboratory for reference analysis.

Duration of treatment / exposure:

98 days

Frequency of treatment:

continuous

Details on study schedule:

The continuous breeding reproduction protocol begins with a 14-day dose range-finding assay (Task 1) that uses small groups of mice. Immediately following dose selection, the continuous breeding phase (Task 2) begins with males and females separated for 1 week. The mating trial begins within a week of the start of treatment and cohabitation continues for 14 weeks to maximize the data collected on fertility. The animals are housed as breeding pairs, one male and one female per cage, for the 14-week period and the offspring of as many as five litters are evaluated during the test. At the end of 14 weeks the male and female are separated and the last litter delivered is used for the second generation.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 1, 2.5, 5% in the feed
Basis:
nominal in diet

No. of animals per sex per dose:

20

Control animals:

yes, concurrent no treatment

Details on study design:

The mice were exposed to the chemical for a 7-day premating period, and were then randomly grouped as mating pairs and cohabited and treated continuously for 98 days. Data were collected on all newborns during this period (body weight, proportion of males’ number of litters per pair, number of live and dead pups) within 12 hr of birth after which each litter was discarded. After the 98-day cohabitation, the pairs were separated but continued on treatments. During the next 21 days, any final litters were delivered and kept for at least 21 days (weaning). For those chemicals for which an offspring assessment of reproductive function was planned (Task 4), the mother was dosed through weaning and F1 mice were dosed until mated at 74 ± 10 days of age. For this, male offspring were mated to female offspring from the same treatment group (n = 20/group/ sex), and the F2 litters were examined for litter size, sex, and pup weight.
When significant adverse effects on fertility were observed in the continuous breeding phase, a crossover mating trial (Task 3) was usually performed to determine whether F0 males or females were more sensitive to the effects. High-dose animals of each sex were mated, to control mice of the opposite sex to determine the affected sex. The high-dose animals were selected to increase the possibility of detecting effects in the crossover mating. There were three combinations of control and treated mice: control males with control females, high-dose males with control females, and control males with high dose females. The offspring of the crossover matings were analyzed as in Task 4 above, and the parents were necropsied. Results of mating high-dose mice with control group partners were compared to matings within the control group to determine which sex was adversely affected. The crossover mating was not done if significant reproductive effects were not observed in the continuous breeding phase.
Task 4 was conducted using F1 animals from the control group and treated animals exposed to the test chemical at the treatment levels indicated.

Positive control:

no data

Examinations

Parental animals: Observations and examinations:

no. litters per pair, no. live pups per pair, no. live male/female pups per litter,sex of pups born alive, mean live pup weight per litter, live female/male pup weight per litter,

Oestrous cyclicity (parental animals):

For the 12 days preceeding necropsy, estrous cyclicity is recorded by taking cell samples by vaginal lavage.

Sperm parameters (parental animals):

At necropsy, the endpoints of male reproductive function included percentage motile sperm, epididymal sperm concentration, and percentage abnormal sperm.

Litter observations:

no. live pups per litter, no.live male/female pups per litter, sex of pups born alive, live pup weight per litter, live male/female pup weight per litter, Adj. live male/female pup weight per litter,

Postmortem examinations (parental animals):

Necropsies were performed in this series of studies, usually on only F0 mice involved in the crossover mating trial, when there was evidence of an effect on reproduction or, at the least, in the second generation even if there was no effect on the F0 mice. Endpoints examined for the females included selected organ weights and histology. At necropsy, the endpoints of male reproductive function included selected organ weights and histology, percentage motile sperm, epididymal sperm concentration, and percentage abnormal sperm (Wyrobek and Bruce, 1975).

Postmortem examinations (offspring):

at the least, in the second generation even if there was no effect on the F0 mice. Endpoints examined for the females included selected organ weights and histology. At necropsy, the endpoints of male reproductive function included selected organ weights and histology, percentage motile sperm, epididymal sperm concentration, and percentage abnormal sperm (Wyrobek and Bruce, 1975).

Statistics:

The Cochran-Armitagc test (Armitage, 1971) was used to test for a dose-related trend in fertility. Pairwise comparisons involving mating and fertility indices were performed using Fisher’s exact test.
The number of litters and the number of live pups per litter were computed on a per fertile pair basis and treat¬ment group means were then determined. The proportion of live pups was defined as the number of live pups divided by the total number of pups. The sex ratio was expressed as the proportion of male pups bom alive to each fertile pair. Dose group means for these parameters were tested for overall differences (Task 3) by using the Kruskal-Wallis test (Conover, 1980) and for ordered differences (Tasks 2 and 4) using Jonckheere's test (Jonckheere, 1954). Pairwise comparisons of treatment group means were performed by applying the Wilcoxon- Mann-Whitney U test.
A Kruskal-Wallis test was also performed on average pup weight‘ Since the number of pups in a litter may affect the average weight of the litter; an analysis of covariance (Neier and Wasserman, 1974) was also used to test for treatment differences in average pup weight, adjusting for average litter size (live and dead pups). Pairwise comparisons were done uang a two-sided t test.
For the organ weights，least-squaixs treatment group means were generated from an analysis of covariance (with body weight as the covariate) and were tested for overall equality using the Ftesi and for pairwise equality using a £ test. All comparisons were two-sided. The Kruskal-Wallis and Wilcoxon-Mann-Whitney U tests were also employed. Historical control data were analyzed and statistical sensitivity was calculated as described by Morrissey et al. (1988b,c).

Reproductive indices:

fertility index, mean no. litters per pair, mean no. live pups per pair, mean no. live male/female pups per litter, proportion of pups born alive, sex of pups born alive, mean live pup weight per litter, mean live female/male pup weight per litter, Adj. mean live male/female pup weight per litter, adjusted mean live pup weight per litter,
A pair was judged to be fertile if it produced one or more litters. A litter was defined as one or more live or dead pups. Fertility index=(No. fertile/No.cohabited) *100.

Offspring viability indices:

mating index, fertility index, mean no. live pups per litter, mean no.live male/female pups per litter, proportion of pups born alive, sex of pups born alive, mean live pup weight per litter, mean live male/female pup weight per litter, Adj. mean live male/female pup weight per litter,

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

not specified

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

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:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

The effects of chemical treatment at the levels specified are reported for the continuous breeding task (Task 2, Table 1) crossover mating trial (Task 3,Table 2) and offspring assessment (Task 4, Table 3). A technical report describing the results of each study is available.® The estimated dose for each task was based on mean body weight awd periodic estimates of feed or water consumption, except for studies conducted by other modes [bisphenol A (subcutaneous implant), dibro-mochloropropane (gavage), and methyl salicylate (gavage)]. Of these 48 studies, adverse effects on fertility or other endpoints in Task 2 or Task 4 (offspring) were detected in 43. Of the 5 negative studies [diethylene glycol monoethyl ether, propylene glycol, triethylene glycol diacetate, di-n-octylphthalate, and trichloroethylene (mouse)], there were 2 (in Task 2) that had adverse effects on pup weight adjusted for litter size. Of the 42 positive studies, there was an adverse effect on one or more parameters in Task 2 other than adjusted pup weight(s)there were often multiple endpoints affected, the least affected endpoints tended to be sex ratio, proportion of pups bom alive, and fertility index (No. fertile/No. cohabited). Among those endpoints that were most often affected, the lowest observed adverse effect level exclusive of reproductive organ weight and sperm measurement data which were previously reported (Morrissey et al 1988b)] was most frequently determined by pup weight, number of pups per litter, or by multiple endpoints (Table 4). This level for all 4 methyl- xanthine studies was determined by a decrease in number of pups per litter (see Morrissey et alr 1988a for discussion). In this first series of 48 studies, reproductive organ weight and sperm measure data were usually collected for only one dose group and the control at the end of Task 3. In most of these RACB studies, adverse effects on fertility in (Task 2) a crossover mating trial was conducted to determine the affected sex (Table 2). In 10 studies both sexes were affected by treatment at the level tested, in 9 studies only females were affected, and in 9 studies the affected sex could not be determined based on fertility (fertility index or number of live pups per litter). Some of the possible explanations for these results will be considered in the Discussion.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

>= 14 400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

Results from a single mating trial using the offspring from the last litter of the continuous breeding task are shown in Table 3; not all studies included this evaluation, and not all dose levels were used when the task was conducted. Only one chemical, di-n-ethyl phthalate, produced an adverse effect on fertility of the offspring in the absence of an adverse effect on the parents. In 13 studies there were adverse effects in both generations. Eight chemicals that affected endpoints other than adjusted pup weight in the continuous breeding phase failed to affect fertility endpoints in the offspring when tested at the same treatment level that caused the adverse effect in Task 2. Five chemicals were nontoxic in both generations (tested at estimated doses of at least 1g/kg/day).
For control pairs of mice involved in the continuous breeding portion of these studies, the fertiJity index was very high, as was the mean number of litters produced per pair (4.7 out of a possible 5.0). Most of the pups were bom alive and were evenly distrib¬uted between the sexes. Male pups were slightly heavier than female pups, and dams weighed more after the final litter than after the first litter was delivered. Survival was excellent among the pups from the last litter (the only one that was kept), and male pups maintained a slight weight advantage through the first 14 days of life. The statistical sensitivity was very high for most of the endpoints in Task 2 , Altemtions of 4 to 7% in below 10%. The fertility index and number of pups per litter, however, were still less sensitive than in the continuous breeding phase and differences of 36 and 19% were necessary to detect significant changes for those endpoints, respectively. This was an improvement compared to the sensitivity of the crossover mating trial (Task 3), but was less powerful than the continuous breeding phase (Task 2).

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 14 400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Results: F2 generation

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

>= 14 400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a 2-generation continuous breeding study in mice, Propylene glycol did not show any adverse effects on parental, F1 and F2 generations at the highest tested dose of 5% in feed (equivalent to 14400 mg/kg/day).

Executive summary:

The purposes of this paper was to summarize the results of 48 continuous breeding studies, evaluate historical control data, compute the statistical power associated with the endpoints, and compare the design and results with multigeneration reproduction studies, where available, on the same chemicals.

These studies were conducted at two different laboratories over a 3-year period. A 14-day dose-setting study (Task 1 utilized one control group and five groups of dosed animals (n = 8 males and 8 females per treatment level). The endpoints for this study were clinical signs, mortality, body weight gain, and consumption of food and water. The continuous breeding phase (Task 2) used a control group (n = 40 animals of each sex) and three treatment groups (n =20 animals of each sex). Of these 48 studies, adverse effects on fertility or other endpoints in Task2 or Task 4 (offspring) were detected in 43. Of the 5 negative studies [diethylene glycol monoethyl ether, propylene glycol, triethyl- ene glycol diacetate, di-octylphthalate, and trichloroethylene (mouse)], there were 2 (in Task 2) that had adverse effects on pup weight adjusted for litter size.

Based on the data as above, for propylene glycol, there was no adverse effects producted for any test parameters at any levels of 0, 2.5 and 5% in feed or water in the studies.