Alcohols, C8-14, γ-ω-perfluoro

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

n.a.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study is a scientific publication and well documented.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Title:

Evaluation of the reproductive and developmental toxicity of a fluoroalkylethanol mixture

Author:

Mylchreest, E., Ladics, G.S., Munley, S.M., Buck, R.C., and Stadler, J.C.

Year:

2005

Bibliographic source:

Drug Chem. Toxicol. 28 (2005): 159-175

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Fluoroalkylethanol mixture

IUPAC Name:

Fluoroalkylethanol mixture

Constituent 2

Reference substance name:

65530-60-1

EC Number:

613-814-1

Cas Number:

65530-60-1

IUPAC Name:

65530-60-1

Test material form:

other: medium tan waxy solid

Details on test material:

- The fluoroalkylethanol [also known as fluorotelomer alcohol, F(CF2CF2)nCH2CH20H, n = 3 to 6] mixture (CAS no. 65530-60-1) as commercially produced was supplied as a pale to medium tan waxy solid by DuPont Chemical Solutions Enterprise (Deepwater, NJ, USA).
- The purity of the test substance was 95%.
- The test substance contained 2% residual fluoroalkylethyl iodide [F(CF2CF2)nCH2CH2I, n =3 to 6], which is the chemical precursor to the fluorotelomer alcohol.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female (nulliparous and nonpregnant) Crl:CD (SD)IGS BR rats were received from Charles River Breeding Laboratories (Raleigh, NC, USA).The rats were approximately 3 weeks old on arrival and approximately 7 weeks old at study start.
Rats were housed one per cage, sexes separate, in stainless steel, wire-mesh cages suspended above cage boards, except when housed as breeding pairs during the cohabitation period. During the gestation period, female rats were housed individually until gestation day (GD) 20.
Beginning on GD 20 for mated females or at the end of the cohabitation period for females without evidence of copulation, female rats were housed individually in polycarbonate pans with bedding. During the lactation period, adult female rats were housed with their litters in polycarbonate litter boxes.
Animal rooms were maintained on a timer-controlled, 12-hour light/12hour dark cycle (fluorescent light). Environmental conditions of the rooms
were targeted to be within a temperature range of 22 ± 3°C and a relative humidity range of 50 ± 20%. Tap water and PMI Nutrition International, Inc., Certified Rodent LabDiet 5002 were available ad libitum.
Initially, all rats were fed pelleted chow; however, beginning on test day 57, animals exhibiting dental problems were placed on ground chow. On test day 74, all rats in the 250 mg/kg/day dose group were placed on ground chow. Animals were humanely cared for and sacrificed according to principles described in Guiding Principles in the Use ofAnimals in Toxicology.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous methylcellulose

Details on exposure:

rats (20 per sex per group) were given dosages of 0, 25, 100, or 250 mg/kg/day for a period of 74 days prior to cohabitation, and during mating, gestation, and lacatation. The fluoroalkylethanol mixture (FTOH mixture) was administered daily by gavage to Sprague-Dawley rats as a suspension in 0.5% aqueous methylcellulose.

Details on mating procedure:

Male and female (nulliparous and nonpregnant) Crl:CD (SD)IGS BR rats were housed one per cage, sexes separate, in stainless steel, wire-mesh cages suspended above cage boards, except when housed as breeding pairs during the cohabitation period.
After approximately 10 weeks of exposure to the test substance, on test day 74, each P1 female was continually housed on a 1:1 basis with a
randomly selected male of the same dose level in the male's cage. On the day copulation was confirmed, the female was transferred back to individual cage housing. Mating pairs were co-housed until evidence of copulation was observed (designated as GD 0), or until 2 weeks elapsed. The presence of an intravaginal or extruded copulation plug was considered evidence of copulation.

Vaginal smears were collected daily beginning 3 weeks prior to cohabitation with males and continuing until copulation was confirmed or the cohabitation. During the gestation period, female rats were housed individually until gestation day (GD) 20. Beginning on GD 20 for mated females or at the end of the cohabitation period for females without evidence of copulation, female rats were housed individually in polycarbonate pans with bedding

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Rats were given dosages of 25, 100, or 250mg/kg bw/d for a period of 74 days prior to cohabitation, and during mating, gestation, and lactation.

Animals were intubated for the 74-day premating period and during the cohabitation (mating) period. P1 male rats continued to be intubated until sacrificed after siring litters (approximately 110 days of exposure). Pregnant female rats were intubated during the gestation period. Lactating females and females with no evidence of copulation were intubated until sacrifice on postpartum day 21 (weaning of the F I offspring).

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 25, 100, or 250 mg/kg bw/d
Basis:
nominal conc.

No. of animals per sex per dose:

Twenty rats per sex were assigned to each dose group

Control animals:

yes, concurrent vehicle

Details on study design:

Body weights, feed consumption, clinical signs, gross pathology, sperm parameters, estrous cyclicity, and reproductive performance were evaluated for the PI generation. The F 1 offspring were evaluated during the lactation period for growth and survival and given a gross pathology examination at weaning. A subset of the offspring were retained; body weights, feed consumption, clinical signs, and age at onset of vaginal opening and preputial separation were evaluated, and gross pathology was performed on postnatal day 60.

Examinations

Parental animals: Observations and examinations:

Rats were weighed once per week during the exposure phase. During the reproduction evaluations, P1 male rats were weighed on a weekly schedule, and P1 female rats were weighed on days 0, 7, 14, 18, and 21 of gestation and lactation. Female rats with no evidence of copulation or that did not deliver a litter were weighed weekly. During the test period, cage-site examinations were conducted at least twice daily.

Feed consumption was measured weekly prior to cohabitation and on GD 0, 7, 14, and 21, but it was not determined during the cohabitation period, for P1 males from the end of cohabitation until sacrifice, or for P 1 females during lactation.

Oestrous cyclicity (parental animals):

Vaginal smears were collected from all P1 female rats in order to evaluate the estrous cycle (% days in estrus, diestrus, proestrus; cycle length). Vaginal smears were collected daily beginning 3 weeks prior to cohabitation with males and continuing until copulation was confirmed or the cohabitation
period ended.

Sperm parameters (parental animals):

Sperm parameters for the first 10 out of 20 P1 males in each exposure group were evaluated. The right cauda epididymis was weighed, and sperm
was collected for evaluation of motility and morphology. The left cauda epididymis and testis were frozen in liquid nitrogen and stored between -65QC and -85QC for the quantitation of homogenization resistant spermatids. Motility and counts were evaluated using the TOX IVOS Sperm Analyzer (Hamilton-Thorne Research, Beverly, MA, USA), and morphology was evaluated using a manual method.

Litter observations:

On PND 0 (day when delivery was complete), 4, 7, 14, and 21, live pups in each litter were counted by sex and individually weighed; pups were individually handled and examined for abnormal behavior and appearance, and any dead, missing, or abnormal pups were recorded. On PND 4, litters were culled randomly to 8 (4 per sex when possible). Litters of 8 pups or less were not reduced. On PND 21, randomly selected weanlings (one
per sex per litter) were placed in individual cages to serve as the F 1 generation but were not dosed.
F 1 generation male and female rats were monitored for vaginal opening (beginning on PND 21) or preputial separation (beginning on PND 35) on a daily basis until criterion was achieved; body weight was recorded on the day of achievement.

Postmortem examinations (parental animals):

P 1 generation female rats were sacrificed on postpartum day 21 and P1 males after siring litters. A gross pathological examination was conducted on all P1 and F1 generation rats. The uteri of all P1 generation females were examined for the presence and number of implantation sites.
The following tissues were saved from P1 and F1 generation rats:
liver (F1 rats only), kidney (F1 rats only), nose (F1 rats only), thyroid (after fixation), pituitary, ovaries, uterus, vagina, testis/testes, epididymis/epididymides, prostate, seminal vesicles, coagulating glands.

Histopathological examination was not conducted for tissue collected from P1 and F1 generation rats, with the exception of reproductive organs of P1 males and females with impaired reproductive performance (those that did not mate or for which copulation did not result in production of a litter). Histopathological examination of reproductive organs was conducted on animals in the subchronic toxicity study (Ladies et al., 2005).

Postmortem examinations (offspring):

F1 generation rats were sacrificed at approximately PND 60. A gross pathological examination was conducted on all P1 and F1 generation rats.
The following tissues were weighed from F1 generation rats:
liver, kidneys, brain, thyroid gland (after fixation), uterus with oviducts and cervix, testes, epididymides, seminal vesicles with coagulating glands, and prostate.
The following tissues were saved from P1 and F1 generation rats:
liver (F1 rats only), kidney (F1 rats only), nose (F1 rats only), thyroid (after fixation), pituitary, ovaries, uterus, vagina, testis/testes, epididymis/epididymides, prostate, seminal vesicles, coagulating glands.

Statistics:

For litter parameters, the litter mean was considered the experimental unit for statistical evaluation (Haseman and Hogan, 1975). For the incidence
of clinical observation, a Cochran-Armitage trend test was applied sequentially (Armitage, 1995; Cochran, 1954).
For the reproduction study, end points measured on a continuous scale (e.g., body weight) and assessed at only one time and only for one sex and where no other factors are important, the data were assessed for monotonicity (Draper and Smith, 1981) in the dose-response, normality (Shapiro and Wilk, 1965), and variance homogeneity (Levene, 1960). Where a monotone dose-response was expected biologically and the data were consistent with that expectation, a step-down (Hochberg and Tamhane, 1987) trend test (Jonckheere, 1954) was applied to determine the no observable effect level (NOEL). Otherwise, pairwise comparison procedures, including Dunnett's test (Dunnett, 1955) for normal, homogeneous data, Tamhane-Dunnett's test (Tamhane, 1979) for normal, heterogeneous data, and Dunn's test (Dunn, 1964) for non-normal data were applied. All tests were applied at the 0.05 significance level. Where body weight effects (vaginal opening, preputial separation) must be considered, a weighted ANCOVA (Analysis of covariance) (Hocking, 1985) was done, and a step-down trend was used based on linear contrasts of least squares means.
Where litter effects (pup/fetal weight, sex ratio) must be considered, a linear contrast of least square means from ANCOVA was sequentially applied;
litter size and sex ratio were considered covariates for pup/fetal weight analyses, and litter size was considered a covariate for sex ratio analysis (Dempster et al., 1984).

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

There were no adverse effects on reproductive parameters observed at 25 mg/kg/d. Implantation sites were comparable in all treatment groups and in comparison with control animals. The number of pregnant animals was not affected by treatment with the test substance. Implantation efficiency however was significantly reduced at 100 and 250 mg/kg/d and reflects a reduction in the number of pups born. On the day of weaning, mean body weights were significantly lower than control in both F1 males and females in the 250 mg/kg/d group. However, mean body weights in these groups progressively returned to control values during the post-weaning period. These findings were considered test-substance related.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No test substance-related mortality occurred in PI or FI generation rats. One death occurred in P1 females in each of the 100 and 250 mg/kg/day groups. One death was the result of dystocia (difficult and prolonged labor) and was not considered test substance-related due to the single occurrence of this finding and the occasional occurrence of this condition in control animals of the strain of rat used in this study. The cause of death was undetermined in the other P1 female but was not considered test substance-related due to the absence of signs of toxicity or gross lesions in that animal. No test substance related clinical signs of toxicity were observed in P1 or F1 generation rats.

There was an increase in tooth clipping required in male rats administered 250 mg kg- I day-I, which was consistent with the observation of dental
problems in the subchronic toxicity study (Ladies et al., 2005).

BODY WEIGHT AND FOOD CONSUMPTION
A statistically significant reduction (89-91% of control mean) in body weight occurred in P1 male rats at 250 mg/kg/day during and after the cohabitation period, but there was no concomitant reduction in body weight gain in P1 males during that period. Statistically significant decrements in body weight, body weight gain, feed consumption (250 mg/kg/day males only), and feed efficiency (males only) occurred in males and females administered 100 and 250 mg/kg/day doses in the reproduction study. After placement of the 250 mg/kg/day animals on ground chow on test day 77, however, an increase in feed consumption, feed efficiency, and body weight gain occurred for both males and females. Therefore, the decrements in body weight and nutritional parameters observed for both males and females appear to be related in part to the test substance-induced alterations to the teeth described above and subsequent decreased ability of the animals to eat the pelleted chow.
Body weight and feed consumption parameters were similar across groups during gestation and lactation in P1 females.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Feed consumption parameters were similar across groups during gestation and lactation in P1 females.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no test substance-related effects on estrous cycle parameters in the P1 generation.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There were no test substance-related effects on sperm morphology, motility, or epididymal sperm counts in the P1 generation. At 100 and 250 mg/kg/day, there was a statistically significant increase in testicular spermatid numbers (123% and 113% of control, respectively) in P1 male rats. This finding was not considered test substance-related because the means for these groups were within the historical control range for previous studies, and the increase appeared to be due to a slightly lower than usual mean in the control group.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating and fertility indices, mean gestation length, and number of implantation sites were comparable across groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
no data
GROSS PATHOLOGY (PARENTAL ANIMALS)
no data

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no toxicologically significant pathology findings in the F 1 pups or P1 or F1 generation adult rats. Histopathological examination was not conducted for tissue collected from P1 and F1 generation rats, with the exception of reproductive organs of P1 males and females with impaired reproductive performance (those that did not mate or for which copulation did not result in production of a litter). Histopathological examination of reproductive organs was conducted on animals in the subchronic toxicity study (Ladies et al., 2005).

OTHER FINDINGS (PARENTAL ANIMALS)
There was an increase in tooth clipping required in male rats administered 250 mg/kg/day, which was consistent with the observation of dental
problems in the subchronic toxicity study (Ladies et al., 2005).

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Results: F1 generation

Details on results (F1)

In the reproduction one-generation study, litter size at birth and number of live pups per litter on day 0 and 4 of lactation, were reduced in groups administered ≥ 100 mg/kg/day. Pup weights during lactation were only significantly reduced at the top dose of 250 mg/kg/d. No other reproductive parameters were affected.
Implantation efficiency however was significantly reduced at 100 and 250 mg/kg/d and reflects a reduction in the number of pups born. At PND 4, the number of live pups was reduced by about 20 % in both treatment groups. The reduction in the number of live pups on PND 0 and 4 was mainly due to three and two litters at 100 and 250 mg/kg/d, respectively, in which loss of several pups or the entire litter was observed early during the lactation period. There were statistically significant reductions in pup weights on lactation days 4, 7, 14 and 21 in the 250 mg/kg/d group.


VIABILITY (OFFSPRING)
At PND 4, the number of live pups was reduced by about 20 % in both treatment groups (i.e. 100 and 250 mg/kg/d). The reduction in the number of live pups on PND 0 and 4 was mainly due to three and two litters at 100 and 250 mg/kg/d, respectively, in which loss of several pups or the entire litter was observed early during the lactation period.
The reduction in implantation efficiency suggests that the reduction in litter size is due to postimplantation embryonic loss or cannibalization of offspring by the dam at parturition. In reproduction studies, where the dam is allowed to deliver her litter, it is often not possible to distinguish between in utero and early neonatal death because of cannibalization of pups by the dam before the pup viability assessment could be performed. However, information on in utero death can be obtained from the standard developmental toxicity study reported in this paper and under Section 7.8.2, in which fetal viability was evaluated by examining the uterine contents just prior to delivery. In the current developmental toxicity study, there was no reduction in the number of live fetuses or increase in resorptions at doses up to 500 mg/kg/day, which suggests that the reduced litter size observed in the reproduction study was not due to in utero death but rather to cannibalization of neonates shortly after birth.

CLINICAL SIGNS (OFFSPRING)
no data
BODY WEIGHT (OFFSPRING)
There were statistically significant reductions in pup weights on lactation days 4, 7, 14 and 21 in the 250 mg/kg/d group.

SEXUAL MATURATION (OFFSPRING)
On PND 4, litters were culled randomly to 8 (4 per sex when possible). Litters of 8 pups or less were not reduced. On PND 21, randomly selected weanlings (one per sex per litter) were placed in individual cages to serve as the F 1 generation but were not dosed. F 1 generation male and female rats were monitored for vaginal opening (beginning on PND 21) or preputial separation (beginning on PND 35) on a daily basis until criterion was achieved.

ORGAN WEIGHTS (OFFSPRING)
no data

GROSS PATHOLOGY (OFFSPRING)
No effects observed

HISTOPATHOLOGY (OFFSPRING)
no data

OTHER FINDINGS (OFFSPRING)

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Executive summary:

In a one-generation reproductive study, rats (20 per sex per group) were given dosages of 0, 25, 100, or 250 mg/kg/day for a period of 74 days prior to cohabitation, and during mating, gestation, and lacatation. The fluoroalkylethanol mixture (FTOH mixture) was administered daily by gavage to Sprague-Dawley rats as a suspension in 0.5% aqueous methylcellulose. All parameters for body weight gain, food consumption, reproductive performance, clinical signs, and developmental landmarks were evaluated as per OECD guidelines.

In the reproduction one-generation study, litter size at birth and number of live pups per litter on day 0 and 4 of lactation, were reduced in groups administered ≥ 100 mg/kg bw/day. Pup weights during lactation were only significantly reduced at the top dose of 250 mg/kg/d. No other reproductive parameters were affected. There were no adverse effects on reproductive parameters observed at 25 mg/kg/d. Implantation sites were comparable in all treatment groups and in comparison with control animals. The number of pregnant animals was not affected by treatment with the test substance. Implantation efficiency however was significantly reduced at 100 and 250 mg/kg/d and reflects a reduction in the number of pups born. At PND 4, the number of live pups was reduced by about 20 % in both treatment groups. The reduction in the number of live pups on PND 0 and 4 was mainly due to three and two litters at 100 and 250 mg/kg/d, respectively, in which loss of several pups or the entire litter was observed early during the lactation period. The authors of the paper discuss the reduction in implantation efficiency might be due to the reduction in litter size which is due to post-implantation embryonic loss or cannibalization of offspring by the dam at parturition. In reproduction studies it is often not possible to distinguish between in utero and early neonatal death because of cannibalization of pups by the dam before the pup viability assessment could be performed. Due to evaluate these effects one should compare results from the developmental study performed in the same rat strain and with the identical substance batch. In the developmental study reported in the same paper, there was no reduction in the number of live fetuses or increase in resorptions at doses up to 500mg/kg /day, which suggests that the reduced litter size observed in the reproduction study was not due to in utero death but rather to cannibalization of neonates shortly after birth. There were statistically significant reductions in pup weights on lactation days 4, 7, 14 and 21 in the 250 mg/kg/d group. On the day of weaning, mean body weights were significantly lower than control in both F1 males and females in the 250 mg/kg/d group. However, mean body weights in these groups progressively returned to control values during the post-weaning period. These findings were considered by the authors test-substance related. 

The NOAEL for the reproductive toxicity parameters evaluated under the conditions of this study was 25 mg/kg/d, based on decreases in the number of pups born, born alive, and pups alive on day 4 of lactation at the 100 mg/kg/d dose level.