DL-hexane-1,2-diol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Reference 1

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.

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

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.

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.

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

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,

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

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

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).

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

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

Based on:

test mat.

Sex:

male/female

Reproductive effects observed:

not specified

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.

Reference 2

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from May 18, 2000 to May 13, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline test following GLP principles

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study) amended by a reproductive assessment

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals and Animal Husbandry
1. Description. Identification and Housing
Young adult, male and female Sprague-Dawley rats were received at the testing facility on May 9, 2000 from Charles River Laboratories, Kingston, NY. Upon receipt, the animals were housed individually in hanging wire cages. Cage cards displaying the pre-randomization/cage number, sex, study number, receipt date and number, facility number, and supplier were affixed to each cage for identification during acclimation and prior to randomization. After randomization, metal ear tags displaying unique identification numbers were used to individually identify the animals, and the animal number, group number, dose level, and treatment group color code was added to each cage card. During the study, immediately prior to and during expanded clinical observation testing sessions, cage cards displaying the blind number only were affixed to each cage. All housing and care was based on the standards recommended by the current Guide for the Care and Use of Laboratory Animals
2. Environment
Environmental control equipment (Edstrom Industries, Environmental Watchdog System, Version 4) was used to monitor/record and adjust environmental conditions as necessary to minimize fluctuations in the animal room environment. The animal room was maintained at room temperature and relative humidity ranges of 74-70 °F and 69-45% t respectively, during the conduct of the study, with the exception of two approximately 30 minute periods on 8/9/00 when the humidity increased to up to 72%. This is a protocol deviation, but it does not affect the integrity of the study. A 12-hour light/12-hour dark cycle was maintained in the animal room during the course of the study, with the exception of occasional <=30 minute disruptions of the light/dark cycle which occurred primarily as a result of collar removal. These disruptions were documented in the facility Edstrom records. In addition, on May 17 and August 23, 2000 the light cycle was disrupted for approximately 2 hours for completion of ophthalmic examinations. Room ventilation was set to produce at least 10 air changes/hour.
Acclimation
Upon receipt, the animals were removed from the shipping cartons, weighed, and placed into individual cages. The animals were examined by qualified personnel and identified with cage cards. Animals were acclimated to the laboratory conditions for seven days. The animals were observed daily for overt physical or behavioral abnormalities, morbidity, and mortality.
Animal Selection
Only healthy animals meeting protocol specified body weight requirements and exhibiting normal behavior were chosen for study use. To avoid potential bias, the animals chosen for study use were randomly selected from all available healthy animals obtained specifically for this study. All animals received a detailed pretest observation period prior to dosing. Females were nulliparous and nonpregnant.
Animals not selected during randomization remained housed in the study room until study day 7. On study day 1, during pre-dosing observations, two of the study animals (G4M2726 and G2F2682) exhibited clinical and dermal, respectively, findings. As a result, these animals were replaced on study with animals not selected previously during randomization. Pretest animal #3 was used to replace G4M2726, and was ear tagged/identified as study animal # G4M2751, Pretest animal # 78 was used to replace G2F2682, and was ear tagged/identified as study animal # G2F2752. On study day 7, animals not assigned to study were physically separated from study animals via transfer to another study and room. The fate of all animals obtained for this study has been documented.

Route of administration:

dermal

Vehicle:

water

Details on exposure:

Vehicle:
Water, treated by reverse osmosis, was used as the vehicle of choice for delivery of the test article.
Dosing
Beginning on study day 1, and for 91-93 consecutive days, the dose solutions were applied daily as a single dermal dose to approximately 10% of the body surface area (BSA) of 15 rats/sex/dose group. For a typical 200-300 g rat, the test site was defined as an approximately 2” x 3" area within the clipped region. The test site was delineated within the clipped area using an indelible marker, and was remarked when necessary.
On study day -1, and as necessary throughout the study, the hair was removed from the dorsal trunk area of the animals using a small animal clipper. The clipped area was approximately 10% of the animal's body surface area, and this area included the scapular (shoulder) region to the wing region of the ileum (hipbone) and halfway down the flank on each side of the animal.
Approximately six hours after the last animal was dosed, the Elizabethan collars were removed from all the animals Dosing was performed sequentially starting with the vehicle control group and proceeding from the low to the high dose groups.
Elizabethan collars were applied to the animal immediately prior to or after application of the dose solution to the test site.The dose solution was administered at a dose volume of 2 ml/Kg using an appropriately sized plastic syringe fitted with a stainless steel ball-tipped. The dose was delivered slowly, spreading evenly over the entire test site during application, using the ball-tipped needle surface and with the aide of a glass rod. Individual doses were initially calculated based on the animal’s day 1 body weight, and doses were adjusted weekly using the most current body weight. Doses administered were based on body weight.
At collar removal, clinical signs observed which were related to the stress of collaring were not documented. Animals scheduled for necropsy on August 29, 30, and 31, 2000 received 91, 92, and 93 doses, respectively. On the last day of dosing for each animal, the Elizabethan collars were removed approximately four hours after the last animal had been dosed.

Details on mating procedure:

none

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose Formulation Analyses
Dose solutions were prepared weekly, and in study weeks 1 through 6, 8 and 13, duplicate samples (5 ml each) were taken from the middle of each concentration of dose solution for analytical purposes. Verification of concentration analysis was performed on samples for use in study weeks 1, 2, 4, 5, 6, 8’ and 13 by Primedica Corporation, Worcester, MA. Samples were taken and sent for analysis from the week 5 dose solution preparation because the mean of the analytical results for one solution prepared week 4 were not within acceptable range (±10% nominal concentration). All samples were sent for concentration analyses on the day of preparation.
Stability and Homogeneity of Dose Formulations
On study day 1, duplicate samples (15 ml each) were taken for stability determination from the middle of both the lowest and highest concentration (175 and 500 mg/ml, respectively) dose solutions prepared. The samples were sent for concentration analyses, to the contact and address listed above, on the day of preparation. Concentration analyses to establish stability of the test article formulations was performed on study days 3 and 9,
On study day 1, duplicate samples (5 ml each) were taken for homogeneity determination from the top, middle, and bottom of each of the lowest and highest concentration (175 and 500 mg/ml, respectively) dose solutions prepared. These samples were shipped as described above. Analyses to establish homogeneity of the test article formulations were performed on study day 3.

Duration of treatment / exposure:

91-93 consecutive days

Frequency of treatment:

once per day

Remarks:

Doses / Concentrations:
0, 350, 700, or 1000 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

Disposition of Test Article
After completion of the proposed studies, remaining test article was returned to the Sponsor Representative. Disposal of all unused portions of the test article solutions was by drain- wasting, according to facility SOPs.
Dose Solution Formulations
The test article was prepared weekly. The test article was prepared as four separate w/v solutions in vehicle, at the following target concentrations for each dose level: 500 mg/ml for the 1000 mg/kg/day level, 350 mg/ml for the 700 mg/kg/day level, 175 mg/ml for the 350 mg/kg/day level, and 0 mg/ml for the vehicle control. For each dose solution, preparation was performed by weighing a specified amount of test article into a clean, tared 500 ml volumetric flask. The exact weight of the test article was recorded. Then reverse osmosis (RO) water was added to the flask to q.s. to the 500 ml volume, and a stir bar was added. The flask was then stoppered, inverted and placed on a magnetic stirrer as necessary to completely mix the components into solution. Solutions, with stir bars, were then transferred to screw cap containers for sampling, storage, and dosing. For the vehicle control dose solution, approximately 500 ml of RO water was added to a clean, capped container. Containers were capped and stored at room temperature when not in use. The test article dose solutions and the vehicle control dose solution (water alone) were delivered daily at a constant dose volume of 2 ml/kg body weight.

study groups design----see table 1 in the field for other informations on method
Rationale for Dosage Level Selection
The dose levels selected by the Sponsor Representative and utilized during the study were
0, 350, 700 and 1000 mg/kg/day. This dose regimen was designed to demonstrate a gradient of limited toxic effects. The high dose was not expected to cause lethality, but to possibly produce signs of toxicity such as decreased body weight. The intermediate dose level was selected for the purposes of evaluating any potential toxicological effects. The low dose was not expected to produce signs of toxicity. The data from this study will be used for human risk assessment.

Positive control:

none

Parental animals: Observations and examinations:

The animals were observed twice daily (a.m. and p.m.) for mortality and moribundity. Body weight, food consumption, and routine detailed clinical observations were recorded weekly. Routine detailed clinical observations, performed in conjunction with body weights, consisted of a detailed examination, including, but not limited to changes in: skin and fur, eyes and mucous membranes, morbidity, posture, respiration, salivation, and behavior Expanded clinical observations were performed in random order weekly, at approximately the same time of day, on the first ten animals/sex/dose group. Expanded clinical observations were performed in a blind fashion and consisted of hand-held and open field observations. During study week 13 expanded clinical observations also included elicited behaviors observations and locomotor activity testing. Ophthalmology examinations were performed prior to study initiation and during study week 13. Estrous cycle evaluations were performed on all female animals for 21 consecutive days, beginning in study week 11 and inclusive through study week 13. After 91-93 days of treatment, the animals were fasted overnight and urine was collected over a period of 16-18 hours overnight prior to necropsy.
In addition, the treated skins of animals in the mid and low dose treatment groups were also processed and evaluated by light microscopy.

Oestrous cyclicity (parental animals):

Estrous cycle evaluations were performed on all female animals for 21 consecutive days, beginning in study week 11 and inclusive through study week 13. Beginning on study day 70 and for 21 consecutive days, estrous cycle evaluations were performed on all female animals. Vaginal smears were prepared and examined daily, and each day's examination was performed within ±2 hours of the previous day's examination if perfonned. Estrous cycle evaluations were completed prior to preparation for the urine collection period before necropsy.

Sperm parameters (parental animals):

After the epididymides had been weighed, the right epididymis was divided in half by cross sectioning in the middle. The head and proximal midsection of the right epididymis were collected in xative for histological preparation and examination. The tail and distal midsection of the right epididymis was placed on dry ice for analysis for total sperm count evaluations. After adequate xation, each testis was sectioned transversely in half and the cranial pole sectioned for routine embedding in parafn, histologic processing and staining with hematcxylin and eosin. The remaining testicular tissue was archived in 70% ethyl alcohol. The left epididymis and the portions of the right epididymis saved for histology were sectioned longitudinally and processed routinely for histologic evaluation. This longitudinal section ensured that the head and mid portion of the epididymides were evaluated histologically. The remaining portions of the epididymides were archived in 10% neutral buffered formalin with the exception of the portions of the right epididymis transferred to Pathology Associates international for sperm analysis. For total sperm count analysis, at necropsy the tail and distal sections of the right epididymis were placed on dry. This frozen section of the epididymis was then transferred to containers and stored frozen at -70 °C until evaluation for total sperm count. Each frozen epididymis was thawed and the caudal section trimmed, weighed, and homogenized into a suspension. A 100 microliter sample of the suspension was treated with a dye which uniquely stains the sperm heads. A sample of the stained sperm was placed into a 20 micron deep glass slide and loaded into the analyzer.
Twenty elds per animal were counted and reported after adjusting for caudal epididymidal weight.
For sperm morphology assessment, two slides of sperm/animal were stained with Eosin. A minimum of 200 sperm cells/animal were evaluated for morphological development.

Litter observations:

none as no multigeneration study

Postmortem examinations (offspring):

none as no multigeneration study

Statistics:

The observed values for body weight, food consumption, clinical pathology parameters, absolute organ weights, grip strength, and Digiscan data were analyzed separately for each sex using either normal distribution or distribution-free techniques. Organ-to-body weight and organ-to-brain weight ratios were calculated and analyzed. If LeveneTs test for homogeneity of variance was not significant (p > 0.05)( comparisons with the control group were made using Dunnett，s multiple comparison procedure. If Levene's test was significant (p £ 0.05), these comparisons were made using a nonparametric analog of Dunnett's procedure. The Dunnett’s procedure and its nonparametric analog fixed the two-sided risk level at a 5% probability that one or more of the comparisons to the control were significant, when in fact a treatment effect does not exist Significance at the 1% and 0.1% levels, and p-values, were also be reported, where appropriate.
Tail flick data was evaluated statistically using a survival analysis method, and the Kaplan-Meier method was used for estimating the 25 ( 50th, and 75th quartile for each group response. This is a protocol deviation, but does not affect the integrity of the study. Pair-wise comparisons between control and treated groups were made using the Wilcoxon test.
The sperm motility, total count, and sperm morphology data were compared across groups using the Kruskal-Wallis nonparametric ANOVA test. If a significant result was obtained at the 5% probability level, the Wilcoxon (Mann Whitney U) test served as the post-hoc group comparison test (at the 5% one-tailed probability level).

Reproductive indices:

The length of an estrus cycle for individual animals was determined by counting the number of days from a recording of estrus (including the first day of estnis) up to (but not including) the next recording of estrus that occurred after metestrus, diestrus,‘ or proestrus were recorded. If estrus was not observed in an apparent cycle, then a metestnis recording that was preceded by either a diestrus or proestrus recording was considered the beginning of the cycle or a diestrus recording that was preceded by a proestrus recording was considered the beginning of the cycle. A full cycle ended with the day prior to a recording of estrus, with the exception that a recording of proestrus on the last (21st) day of estrus evaluations was considered to end a full cycle and this last proestrus recording was included as the nal day of the last estrus cycle. The mean estnis cycle duration (i.e., number of days) was calculated for each animal. Group means and standard deviations for estrus cycle duration were calculated and reported. For estrus cycle evaluations, the total number of days in each phase, thenumber of combined days in proestrus and estrus, and the number of combined days in metestrus and diestrus were calculated for each animal. Group means and . standard deviations for these parameters were calculated and reported.
The sperm motility, total count, and sperm morphology data were compared across groups using the Kruskal-Wallis nonparametric ANOVA test. if a signicant result was obtained at the 5% probability level, the Wilcoxon (Mann Whitney U) test served as the post-hoc group comparison test (at the 5% one-tailed probability level).

Offspring viability indices:

none as no multigeneration study

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

one control male that was euthanized for humane reasons on study day 14. Treatment-related clinical findings included rough coat and fur staining.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The study animals in the 1000 mg/kg/day treatment group exhibited statistically significant decreases in body weight gains per day and in cumulative absolute body weight gains.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The study animals in the 1000 mg/kg/day treatment group exhibited statistically significant decreases in body weight gains per day and in cumulative absolute body weight gains.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic evaluations of tissues from surviving animals indicated that treatment-related changes were restricted to epidermal hyperplasia and hyperkeratosis in treated skin.

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not examined

Dose descriptor:

NOAEL

Effect level:

700 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: Generation: no multigeneration study (migrated information)

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: Generation: no multigeneration study (migrated information)

Dose descriptor:

other: NOAEL for estrous cycle interference

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: Generation: no multigeneration study (migrated information)

Dose descriptor:

other: NOAEL for male reproductive assessment

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: Generation: no multigeneration study (migrated information)

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Reproductive effects observed:

not specified

Result tables of estrous cycle and sperm assessment see attachment in section "Attached background material"

Conclusions:

In this subchronic dermal toxicity study no effects on estrous cycle of female and sperm obility and sperm count of males has been observed up to the highest tested dose of 1000 mg/kg bw/day and thus the substance is considered not to effect fertility in this study.

Executive summary:

Vaginal cytology demonstrated that the estrous cycle data for treated females was comparable to control females. In addition, sperm analysis results demonstrated that there were no statistically signicant differences between treated and control males for percent sperm motility, total sperm count, or percent abnormal sperm morphology. These reproductive assessment results indicate that there were no biologically relevant differences between treated and control animals in this study when dosed up to 1000 mg/kg bw/day by dermal application.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

chronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 µg/kg bw/day

Study duration:

subchronic

Species:

rat

Additional information

A two-generation study reported by Morrissey et al. demonstrated that 1,2-propylene glycol, a structural homologue to 1,2-hexanediol, did not show any effects on fertility when dosed at 1, 2.5 and 5% in diet, had no effect on mean number of litters, life pups, and sex of pups. In their study, Morrissey et al. investigated 48 different substances with several of them having effects on fertility index, litter and pups thus showing the sensitivity of method applied. The highest dose applied (5%) corresponds to 14.400 mg/kg bw/d in mice and was considered as NOAEL for parental, F1 and F2 generation in this study. The read-across justification is presented below.

A 90-day repeated dose toxicity study amended by a reproductive fertility assessment with vaginal cytology on hexane-1,2-diol demonstrated that the estrous cycle data for treated females was comparable to control females. In addition, sperm analysis results demonstrated that there were no statistically significant differences between treated and control males for percent sperm motility, total sperm count, or percent abnormal sperm morphology. These reproductive assessment results indicate that there were no biologically relevant differences between treated and control animals in this study when dosed up to 1000 mg/kg bw/day by dermal application.

Thus, it can be summarized that no effects on fertility through 1,2-hexanediol were observed by dermal exposure and results on 1,2-propylene glycol indicate that even at doses of 5% in diet effects on fertility of mice were not apparent for 1,2-propylene glycol and no further data are required on fertility for this substance.

Hypothesis for the analogue approach from propane-1,2-diol to hexane-1,2-diol

Both, propane-1,2-diol and hexane-1,2-diol are two corresponding members of a homologue series of linear 1,2-alkyldiols, starting with 1,2-propandiol, 1,2-butandiol, 1,2-pentanediol, 1,2-hexanediol etc. Based on very similar physico-chemical properties, common functional groups (vicinal diol) and common toxicokinetik behavior, data for 1,2-propanediol on reproductive toxicity can be used as surrogate data for 1,2-hexanediol.

Source Chemical and Target chemical

	Propane-1,2-diol (data as shown in dissemination tool for REACH registration dossier on Propane-1,2-diol)	Hexane-1,2-diol
CAS No	57-55-6	6920-22-5
EC No	200-338-0	230-029-6
Mol. weight	76.10	118.18
Smiles code	OCC(O)C	OCC(O)CCCC
Appearance	Clear colourless liquid	Clear colourless liquid
Water solubility	Fully miscible (100%)	Fully miscible (>9 g/g)
logPOW	-1.07	0.58
Vapour pressure	20 Pa at 20 °C	0.58 Pa at 20 °C
Melting point	< -20 °C	2 °C
Boiling point	184 °C at 100.32 kPa	228.3 °C
Density	1.03 g/cm3 at 20 °C	0.95 g/cm3 at 20 °C
Purity	Typically >99 %	Typically >99 %
Acute oral toxicity	LD50 (rat) 22000 mg/kg bw	LD50 (rat) >5000 mg/kg bw
Acute dermal toxicity	LD50 (rat) >2000 mg/kg bw	Gap, read across from 1,2-pentanediol LD50 (rat) >2000 mg/kg bw
Acute inhal. toxicity	LC50 (rabbit) >317042 mg/m³air	Gap, read across from 1,2-pentanediol LC50 (rat) >7015 mg/m³air
Corrosion/Irritation	Skin non-irritating Eye non-irritating	Skin non-irritating Eye irritating
Skin sensitization	Not sensitising	Not sensitising
Repeated dose toxicity	Oral rat NOAEL(2a) 1700 mg/kg bw/d male and 2100 mg/kg bw/d female Inhal. rat NOAEC(90d) 1000 mg/ m³air female and 2200 mg/ m³air male	Oral NOAEL(14d) 500 mg/kg by/d Dermal NOAEL(90d) 700 mg/kg bw/d
Genetic toxicity	Negative in in vitro Ames (+/-) in vitro Chrom. Ab. (+/-) in vivo Chrom. Ab. Mouse lymphoma (L5178Y) (+/-)	Negative in in vitro Ames (+/-) in vitro Chrom. Ab. (+/-) in vitro Mouse HPRT (V79) (+/-)
Carcinogenicity	Oral rat NOAEL(2a) 1700 mg/kg bw/d male and 2100 mg/kg bw/d female non-carcinogenic Oral dog NOAEL (2a) 5000 mg/kg bw/d carcinogenicity and 2000 mg/kg bw/d general toxicity non-carcinogenic	No data
Reproductive toxicity	NOAEL (OECD416) 14400 mg/kg bw/d (highest tested dose) for P, F1 and F2 NOAEL(OECD414) 10400 mg/kg bw/d developmental toxicity	NOAEL (OECD414) 300 mg/kg bw/d (highest tested dose) NOAEL(OECD416) gap

Purity / Impurities

Read across is possible provided that there is no impact of impurities on the toxicological properties of the target and source chemicals. For both, impurities are comparable or not present.

Analogue Approach Justification

Both, propane-1,2-diol and hexane-1-2-diol are two corresponding members of a homologue series of linear 1,2-alkyldiols, starting with 1,2-propandiol, 1,2-butandiol, 1,2-pentanediol, 1,2-hexanediol etc. 

Both substances do show similar physico-chemical properties (see above) in particular regards water solubility (fully miscible with water) and low log POW values; consequently, similar oral and dermal absorption is assumed whereas inhalative exposure and absorption is unlikely due to the low vapour pressure of both.

Sharing the same functional group(s) being an alkyl chain with a primary alcohol function and directly associated a secondary alcoholic function (vicinal diol, glycol) following absorption and distribution through blood within the body occurs rapidly and both do share a common metabolism starting with oxidation by alcohol dehydrogenase and thereafter either by further alcohol dehydrogenase to glyoxal derivatives or by aldehyde dehydrogenase to lactates (major pathway). Distribution and following efficient de-toxification pathways via urine as lactates or their glucuronides excretion also is efficient. Bioaccumulation in the body therefore is not apparent (see toxicokinetic assessment in this dossier).

When comparing available toxicological data (see matrix above) acute and repeat toxicity data are in the same high dose range and no significant differences are seen as expected.

Conclusion on bioavailability and metabolism of source and target substance

Both, 1,2-propanediol and 1,2-hexanediol are well absorbed, rapidly distributed and efficiently metabolized to become excreted as glucuronides mainly via urine. Thus, both substances show a common ADME-pattern as expected. This is supported by comparable acute and repeat dose toxicity data. In addition, genetic toxicity and reproductive toxicity do not show and indications for concern (see matrix above). As a consequence it can be reasonably assumed that data on reproductive toxicity on propane-1,2-diol (2-generation study according to OECD 416) can be used to predict reproductive toxicity of hexane-1,2-diol and such read-across approach is valid.

Conclusion on Classification and Labelling / Risk and Hazard Assessment

Data for reproductive toxicity of propane-1,2-diol used are reliable with restriction (Klimisch 2) as they were performed according to OECD 416, according to GLP standards fulfilling basic scientific principles. The results are adequate for deciding upon classification and labeling as well as for concluding upon hazard and risk assessment for reproductive toxicity, since at the highest tested concentration no effects in any generation (P, F1 or F2) was observed and the doses tested were as high as 14400 mg/kg bw/d.

Short description of key information:
The reproductive assessment results on estrous cycle effects and effects of sperm count and mobility indicate that there were no biologically relevant differences between treated and control animals in this study when dosed up to 1000 mg/kg bw/day by dermal application.

Justification for selection of Effect on fertility via oral route:
2-generation reproductive toxicity study with investigation on fertility parameters performed on 1,2-propane diol, used for read-across to the structural analogue 1,2-hexane diol.

Justification for selection of Effect on fertility via dermal route:
One dermal fertility study available

Effects on developmental toxicity

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from January 18,2006 to May 10, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline test following GLP regulations

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

Animals and Animal Husbandly:
A total of ninety-six time-mated female Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches of forty-eight animals two days apart; each delivery batch contained twenty-four animals at Day 1 of gestation and twenty-four animals at Day 0 of gestation. All females were supplied with stud male identification records and were identified using the supplier’s standard identification methods.
Throughout the study the animals were given pelleted diet (Certified Rodent diet PMI 5002 supplied by BCM IPS Ltd., London UK) and water was supplied via bottles attached to each cage, ad libitum. The diet and water were considered not to have contained any contaminants at a level which may have affected the outcome of this study.
Upon arrival the females were temporarily housed in groups of four or five in polypropylene cages with stainless steel grid floors and tops. Animals were then weighed and allocated to the study. Following allocation the females were caged individually in polypropylene cages with solid floors and stainless steel grid tops. Softwood chips were used as bedding material.
The animals were housed in a single air-conditioned room, providing at least fifteen air changes per hour. The room was maintained to operate within a temperature range of 19 and 23 °C and a relative humidity of 40 and 70% with these conditions monitored on a daily basis. The lighting within the room was controlled to allow twelve hours of continuous light within a twenty-four hour period.
Allocation:
On the day of arrival the females were assigned to treatment groups using a randomisation procedure based on stratified bodyweight to ensure, as far as possible, similar group mean bodyweights for each treatment group. The females were assigned to positions on the cage battery using a randomised block design.
At allocation females were given a unique earmark for the study according to the assigned number for each dose group. Each cage was identified with a colour coded cage card containing information including project number, dose group and animal number.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Experimental Preparation
The test material was prepared weekly as a solution in distilled water (vehicle) by weighing an appropriate amount of test material into a suitable container and adding sufficient vehicle to make the required volume. The test material formulations were shaken until all the test material dissolved. Formulations were therefore prepared weekly and stored at approximately +4 °C in the dark.

Dosing
Animals were dosed with the appropriate concentration of dose formulation once daily, using a metal catheter and syringe between Days 5 and 19 of gestation. Control females received vehicle only. The dose administered was adjusted for most recent bodyweight using a constant dose volume of 5 ml/kg bodyweight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test material formulations were determined by Safepharm Analytical Laboratory. Results showed the formulations to be stable for at least fourteen days.

Details on mating procedure:

The day that positive evidence of mating was observed was designated Day 0 of gestation.

Duration of treatment / exposure:

14 days

Frequency of treatment:

once daily

Duration of test:

20 days

Remarks:

Doses / Concentrations:
0 (control), 30, 100, 300 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

Selection of Dose Levels
Dose levels of 300, 100 and 30 mg/kg/day were selected for use on this study based on results of a preliminary oral gavage prenatal developmental toxicity study in the rat (SPL Project Number 2082/0014). In this preliminary study, a dose level of 500 mg/kg/day was associated with adverse clinical signs that excluded this dosage level from use in this main investigation. A high dosage of 300 mg/kg/day was therefore chosen in anticipation of a degree of toxicity that would not impair the assessment of embryofoetal development. Lower dosages represent approximately three-fold reductions from this high dosage

Maternal examinations:

1. Morbidity/Mortality
All females were checked twice daily during the normal working week and once daily at weekends.
2. Clinical Observations
All females were observed once daily, in the morning throughout gestation and, additionally, one hour after dosing, throughout the dosing period, for clinical signs of toxicity.
3. Bodyweight
All females were weighed on Days 3, 5, 6, 7, 8, 11, 14, 17 and 20 of gestation.
4. Food Consumption
Food consumption for individual animals was recorded for discrete periods throughout the study on Day 3 to 5 Day 5 to 8 Day 8 to 11 (or 9 to 11) Day 11 to 14, Day 14 to 17 and Day 17 to 20 of gestation.

Ovaries and uterine content:

Females were killed on Day 20 of gestation by carbon dioxide asphyxiation followed by cervical dislocation. Each animal was examined externally and internally for macroscopic abnormalities. The ovaries and uteri of pregnant females were removed, examined and the following data recorded;
i) Pregnancy status
ii) Number of corpora lutea
iii) Gravid Uterus weight
iv) Number, position and type of intra-uterine implantation
Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue.
Late Death: Separate embryonic/foetal and placental tissue visible.
Dead Foetus: A foetus that had died shortly before necropsy. These were included as late deaths for reporting purposes.

Fetal examinations:

The foetuses were killed by subcutaneous injection of sodium pentobarbitone. Except where conditions dictate otherwise, alternative foetuses were identified using an indelible marker and placed in Bouin’s fixative. These foetuses were transferred to 90% industrial methylated spirits (IMS) in distilled water and examined for visceral anomalies under a low power binocular microscope. The remaining foetuses were identified using colour coded wires and placed in 70% IMS in distilled water. The foetuses were eviscerated, processed and the skeletons stained with alizarin red. The foetuses were examined for skeletal development and anomalies.
All implantations and viable foetus were numbered according to their intra-uterine position as follows (V = viable foetuses):
Left Horn Cervix Right Horn
L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
VI V2 V3 V4 V5 V6 V7 V8 V9 V10 Vll V12 V13 V14 V15 V16
v) Foetal sex
vi) External foetal appearance
vii) Foetal weight
viii) Placental weight

Statistics:

Data were processed to give litter mean values, group mean values and standard deviations (where appropriate). The litter was regarded as the standard unit of assessment therefore values were first calculated within each litter and group mean values were calculated as the mean of these litter values.
The following parameters were analysed statistically, where appropriate, using the test methods outlined below:
Bodyweight, bodyweight change and food consumption: Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnet’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.
Litter data and litter, placental and foetal weights: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control value against treated values.

Indices:

Percentage pre-implantation loss was calculated as:
(Number of corpora lutea - number of implantations)/ number of corpora lutea
Percentage post-implantation loss was calculated as:
(Number of implantations - number of live foetuses)/ number of implantations

Historical control data:

no data

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
1 Clinical Observations and Mortality
There were no clinical signs associated with treatment and all females survived to scheduled termination on Day 20 of gestation.
2 Bodyweight
No adverse effects of treatment on bodyweight or bodyweight gain were apparent throughout the study at 300, 100 or 30 mg/kg/day
3 Food Consumption
No adverse effects of treatment on food consumption were apparent throughout the study at 300, 100 or 30 mg/kg/day,
4 Necropsy
All animals were pregnant and no macroscopic abnormalities were observed at macroscopic post mortem examination on Day 20 of gestation.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: other:

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter Data and Litter Placental and Foetal Weights
There was no obvious adverse effect of maternal treatment on in-utero offspring survival, as assessed by the mean numbers of early or late resorptions, live litter size and post-implantation loss at 300, 100 or 30 mg/kg/day. Sex ratio was essentially similar in all groups and did not indicate any selective effect on survival for either sex.
Intergroup differences in mean litter, placental and foetal weights did not indicate any obvious effect on embryofoetal growth at these dosages.
2. Foetal examination
Neither the type, distribution nor incidence of foetal findings observed during external examination at necropsy or subsequent detailed visceral and skeletal examinations indicated any adverse effect of maternal treatment on foetal growth or development at 300, 100 or 30 mg/kg/day.

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Based on the results given in this study, test article caused no adverse effect on the pregnant rat on the developing conceptus at dose levels up to 300 mg/kg. The ‘no-observed adverse effect level’(NOAEL) was therefore considered to be 300 mg/kg/day.

Executive summary:

This study was conducted following OECD guideline 414 and GLP regulations to investigate the effects of test article on the embryotoxic/teratogenic development of rat. A total of 96 time-mated female Crl:CD(SD) IGS BR strain rats were administered orally, once daily, by gavage, at dose levels of 0, 30, 100 and 300 mg/kg/day. Clinical signs, bodyweights and food consumption were recorded during the study. The females were killed on Day 20 of gestation and subjected to macroscopic necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, foetal weight, sex and external and internal macroscopic appearance were recorded. Treatment at dosage up to 300 mg/kg/day was well tolerated by the pregnant females and was not associated with any effects on clinical condition, bodyweight change, food intake or necropsy observations. There were no effects at dosages up to 300 mg/kg/day on embryofoetal survival, growth and development.

The 'No- observed-adverse-effect-level' (NOAEL) for the pregnant female and for embryofoetal survival, growth and development was therefore considered to be 300 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental toxicity

A study was conducted following OECD guideline 414 and GLP regulations to investigate the effects of test article on the embryotoxic/teratogenic development of rat. A total of 96 time-mated female Crl:CD(SD) IGS BR strain rats were administered orally, once daily, by gavage, at dose levels of 0, 30, 100 and 300 mg/kg/day. Clinical signs, bodyweights and food consumption were recorded during the study. The females were killed on Day 20 of gestation and subjected to macroscopic necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, foetal weight, sex and external and internal macroscopic appearance were recorded. Treatment at dosage up to 300 mg/kg/day was well tolerated by the pregnant females and was not associated with any effects on clinical condition, bodyweight change, food intake or necropsy observations. There were no effects at dosages up to 300 mg/kg/day on embryofoetal survival, growth and development.

The 'No- observed-adverse-effect-level' (NOAEL) for the pregnant female and for embryofoetal survival, growth and development was therefore considered to be 300 mg/kg/day (highest dose applied).

In a supportive OECD 414 study even higher doses were tested and this study also did not show any significant effects on developmental toxicity at the highest dose group (750 mg/kg bw/d) in this study which was set as NOAEL for developmental toxicity. Effects seen at the highest dose group on maternal body weight gain and reduced feed consumption lead to a NOAEL for maternal toxicity of 500 mg/kg bw/d in this study. Thus, it is apparent that 1,2-hexanediol does not show developmental toxicity in these 2 studies, even at doses showing slight signs of maternal toxicity.

Justification for selection of Effect on developmental toxicity: via oral route:
A guideline and GLP compliant developmental toxicity study in rats is available.

Justification for classification or non-classification

1,2-Hexandiol does not have to be classified regarding toxicity to reproduction according to the criteria laid down in the EU Dangerous Substances Directive (67/548/EEC) and in the EU Classification Labelling and Packaging Regulation (1272/2008/EC), because

- 1,2-Hexandiol caused no developmental toxic effects in a OECD 414 and GLP compliant oral gavage study in rats at the highest tested dose of 300 mg/kg/day, and

- 1,2-Hexandiol caused no effects on estrous cycle of female and sperm mobility and sperm count of males or macroscopic or microscopic effects on the reproductive organs in a dermal subchronic toxicity study at the highest tested dose of 1000 mg/kg/day, and

- in a 2-generation continuous breeding study in mice, propylene glycol (read-across substance to 1,2-Hexandiol) 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).