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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

One gereration reproductive toxicity, oral (OECD 415, read across), rat:

NOAEL (fertility) = 500 mg/kg bw/day

NOAEL (systemic, male) = 100 mg/kg bw/day

NOAEL (systemic, female) = 500 mg/kg bw/day

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across: supporting information
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects on parental fertility observed at highest dose tested.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse effects observed at 100 mg/kg bw/day.
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects observed up to the highest dose tested.
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Remarks:
development
Generation:
F1
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed on development at 500 mg/kg bw/day.
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
An analogue substance approach was used for read-across to the target substance ammonium undecafluorohexanoate (CAS 21615-47-4). In a combined subchronic repeated dose oral toxicity study with 1-generation reproduction study in rats with source substance sodium perfluorohexanoate (CAS 2923-26-4), the NOAEL for systemic toxicity was considered to be 500 mg/kg bw/day for P0 females and 100 mg/kg bw/day for P0 males based on reduced body weight and body weight weight gain in males at 500 mg/kg bw/day. No adverse effects on parental fertility occurred up to the highest dose tested, therefore the NOAEL for fertility was 500 mg/kg bw/day. A transient reduction in body weight and body weight gain was observed in male and female F1 rats at 500 mg/kg bw/day during lactation period and first weeks post-weaning. These effects were considered not to be toxicologically relevant since they were only transient. Therefore, the NOAEL for developmental toxicity was considered to be 500 mg/kg bw/day. Sodium perfluorohexanoate is therefore concluded not to present a reproductive or developmental hazard and no classification is justified.
Based on the analogue approach, no toxicity to reproduction is expected for the target substance.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study from a reference substance with similar structure and intrinsic properties. Read-across is justified based on common precursors and breakdown products of hydrolysis and consistent trends in environmental fate, ecotoxicological and toxicological profile (refer to endpoint discussion for further details). The selected to study is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Justification for read-across

There is no data available on the fertility of ammonium undecafluorohexanoate. The assessment of developmental/reproductive toxicity was  based on a study conducted with a suitable source substance as part of a read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

Fertility

CAS 2923-26-4

Sodium perfluorohexanoate (CAS 2923-26-4) was tested in a subchronic repeated dose oral toxicity study with one-generation reproduction study according to OECD Guideline 408 and 415 and in compliance with GLP (Chemours, 2009). 20Crl:CD(SD) rats/sex/group were administered the test substance via gavage at dose levels of 0, 20, 100, or 500 mg/kg bw/day. The control group received the vehicle (water) only. Administration to males and females occurred during 70 days of premating and continued during 2 weeks mating period. In dams treatment was continued throughout gestation and lactation period until the day the pups were weaned (postpartum day 21), for a total of approx. 126 days. Males were treated throughout the habitation period until the day before sacrifice for a total of approx. 110 days.

The P0 animals were observed for mortality and the presence of clinical signs daily. Detailed clinical observations were made weekly, and body weight and food consumption were determined at least weekly throughout the study for P0 rats. Estrous cycle parameters (percent days in diestrus, proestrus, and estrus) and estrous cycle length were determined for 3 weeks prior to cohabitation and up to the day before evidence of copulation was observed in P0 females. The number of uterine implantation sites was recorded for all P0 adult females. Selected organs, including reproductive organs, were collected and saved from P0 adults. Reproductive organs from P0 rats with suspected reduced fertility were examined microscopically.

Sperm motility, morphology, and concentration in the cauda epididymis, and spermatid concentration in the testis were determined for the first 10 surviving parentalmale rats on each treatment level. P0 animals were sacrificed as follows: on Day 21 of lactation for P0 females, on test days 111 for P0 males. A gross pathology examination was performed on all P0 adults. 

Litter examinations (number of live and dead, individual pup weights, clinical observations) were determined at birth, and on Days 4, 7, 14 and 21 of lactation. The litter size was standardised on postnatal day (PND) 4. The age and body weight at vaginal opening or preputial separation were recorded for the F1 generation. On PND 21, 3 F1 weanlings/sex/litter, litter size permitting, were sacrificed. The remaining F1 males and females were sacrificed on PND 60.

The F1 animals were observed for mortality and the presence of clinical signs daily. Detailed clinical observations were made weekly, and body weight and food consumption were determined at least weekly throughout the study for adult F1 rats. A gross pathology examination was performed on all F1 weanlings sacrificed on PND 21 and on F1 adults. Selected organs, including reproductive organs, were collected and saved from F1 adults. 

P0 animals:

There was no test substance-related mortality in P0 adult rats at any dose level. Clinical signs of increased incidence of stained skin/fur in P0 males and females were observed at 500 mg/kg bw/day. Significantly lower body weight (>10%) compared with the control were observed in males at 500 mg/kg bw/day. In this group the body weight was 7-15% lower than control on test days 49-105; contributing to an overall body weight gain reduction (29%) as compared with the control group. In females of 500 mg/kg bw/day group, a reduction in body weight gain (29%) compared with the control was observed during first week of gestation period, but was transient and therefore not toxicologically relevant. The females gained additional weight during the lactation period.

The food consumption was reduced (11% lower than control) in females at 500 mg/kg bw/day during the first week of gestation. As a consequence, food efficiency was also reduced (19% lower than control) in this period. In males, overall food efficiency was reduced (24% lower than control) at 500 mg/kg bw/day compared with the control as a result of reduced body weight gain throughout the premating period.

There were no effects considered to be related to test substance treatment at any dose level on mating and fertility index, gestation length, number of implantation sites, implantation efficiency, pup survival, and estrous cycle parameters in P0 female rats. Sperm parameters were comparable across dose groups in P0 male rats.

Under the conditions of this study, the NOAEL for systemic toxicity was considered to be 500 mg/kg bw/day for females and 100 mg/kg bw/day for males based on lower body weight and body weight gain in males at 500 mg/kg bw/day, compared with the control group. No adverse effects on parental fertility occurred up to the highest dose tested, therefore the NOAEL for fertility was 500 mg/kg bw/day.

F1 animals:

Lactation period, PND 0-21:

There were no significant differences between the control group and treatment groups on pup survival up to PND 21, sex ratio, clinical signs, and day of vaginal opening or preputial separation.

Significant reductions in pup weights were observed (17-18% lower than control group) at 500 mg/kg/day during PND 0-21.

 

Post-weaning period:

No treatment-related mortality or clinical signs were observed in the F1 generation at any dose level. In males at 500 mg/kg bw/day, food consumption was statistically significantly reduced (13% lower than control group) during the first week after weaning.

Body weight, males: Body weights were significantly reduced compared with the control group throughout the post-weaning period at 500 mg/kg bw/day. This was due to a lower body weight of the high-dose group on Day 0 post-weaning; the body weight gain of the high-dose pups was comparable to the control group. During the first 4 weeks post-weaning, the body weights were 7-16% lower than control group. Body weight gain during the first week after weaning was 16% lower than control group at 500 mg/kg bw/day and the difference was reduced over the following three weeks. Body weight of this group was similar to the control group from Day 28 after weaning. Overall body weight gain (post-weaning days 0-39) was comparable across dose levels.

Body weight, females: Body weights were reduced (11%-12% lower than control group) during the first week (post weaning) at 500 mg/kg bw/day. Body weight of this group was similar to the control group from Day 14 after weaning. There were no test substance-related effects on body weight gains in F1 adult female rats at any level tested.

In conclusion, the differences in body weight and body weight gain observed between the high-dose group and control group are not considered to be toxicologically relevant, since the effects were transient. During the postweaning period, the high-dose offspring increased the body weight gain until the body weights were comparable with the control.

There were no test substance related pathology findings at any dose level in the F1 generation.Sexual maturation was unaffected up to the highest dose tested and no treatment-related effects on development and teratogenicity in F1 male and female rats were observed.

 

The NOAEL for developmental toxicity was considered to be 500 mg/kg bw/day. Sodium perfluorohexanoate is therefore concluded not to present a reproductive or developmental hazard and no classification is warranted.

Effects on developmental toxicity

Description of key information

Prenatal developmental toxicity study (OECD 414, read across), rat: NOAEL (development) = 100 mg/kg bw/day

Postnatal developmental toxicity study (no guideline followed), mouse: maternal NOAEL = 175 mg/kg bw/day; developmental NOAEL = 175 mg/kg bw/day

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No maternal adverse systemic effects observed at 100 mg/kg bw/day.
Remarks on result:
other: Source: CAS 2923-26-4, Chemours, 2007
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: Source: CAS 2923-26-4, Chemours, 2007
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed at the highest dose tested.
Key result
Abnormalities:
no effects observed
Key result
Dose descriptor:
NOAEL
Remarks:
development
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No fetal adverse effects on development observed at 100 mg/kg bw/day.
Remarks on result:
other: Source: CAS 2923-26-4, Chemours, 2007
Dose descriptor:
LOAEL
Remarks:
development
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Remarks on result:
other: Source: CAS 2923-26-4, Chemours, 2007
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
An analogue substance approach was used for read-across to the target substance ammonium undecafluorohexanoate (CAS 21615-47-4). In a developmental toxicity study in rats with source substance sodium perfluorohexanoate (CAS 2923-26-4), the NOAEL for maternal systemic toxicity was 100 mg/kg bw/day, based on reductions in maternal body weight parameters and food consumption. The NOAEL for developmental toxicity was 100 mg/kg bw/day based on decreased fetal weights at 500 mg/kg bw/day. Since reduced fetal weights are assumed to be a secondary effect of maternal systemic toxicity at the same dose level of 500 mg/kg bw/day, sodium perfluorohexanoate is therefore concluded not to present a developmental hazard.
Based on the analogue approach, no toxic effects on development are expected for the target substance.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Dec 2009 - 26 Feb 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Maternal treatment was only from day 6 to 18 post coitum.
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: In this combined developmental and perinatal/postnatal reproduction study toxic effects/disturbances resulting from test substance treatment of Crl:CD1(ICR) pregnant female mice and development of the embryo and fetus consequent to exposure of the dam from implantation to closure of the hard palate and during lactation was evaluated. This study was designed to evaluate the ICH Harmonised Tripartite Guideline stages C through F of the reproductive process and detect effects on gestation, parturition, lactation and maternal behavior in female mice, and on the development of the offspring of the treated female mice. Because manifestations of effects induced during this period may be delayed in the offspring, observations were continued through sexual maturity of the F1 generation mice.
- Short description of test conditions: Eighty presumed pregnant Crl:CD1(ICR) mice were randomly assigned to four dosage groups, 20 mice per group. Solutions of the test substance and/or the vehicle were administered orally once daily from Day 6 of gestation (GD 6) through GD 18. After completion of the 20 day postpartum period (PPD 20), F0 generation female mice were sacrificed and liver samples were collected from 5 mice per group for pharmacokinetic analysis; mice that did not deliver a litter were sacrificed on GD 23. Additionally, on PPD 20, all pups not selected for continued evaluation were sacrificed. F1 generation mice selected for continued evaluation were sacrificed on PPD 41. Blood and liver samples were collected from five mice per sex per group for pharmacokinetic analysis.
- Parameters analysed / observed: The following parameters were evaluated for F0 generation female mice: viability, clinical observations, body weights, body weight changes, maternal behavior, litter observations, natural delivery, pup body weights, dam and pup necropsy observations. The following parameters were evaluated for F1 generation male and female mice: viability, clinical observations, body weights, body weight changes, eye opening, age of sexual maturity and necropsy observations.

GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
other: Crl:CD1(ICR)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., St Constant, Canada
- Age at study initiation: approx. 63 days at day of arrival
- Weight at study initiation: 25.8 - 31.7 g (range)
- Housing: F0 was housed individually in stainless steel, wire bottom cages, except during the cohabitation and postpartum periods. During cohabitation, each pair of male and female mice was housed in the male mouse’s cage. Each dam and delivered litter was housed in a common nesting box with nesting material (Bed-o´cobs bedding) during the postpartum period. F1 was housed paired in nesting boxes until PND 27, afterwards individually.
- Diet (e.g. ad libitum): Certified Rodent Diet #5002 (PMI Nutrition International, Inc., St. Louis, MO, USA), ad libitum
- Water (e.g. ad libitum): Local water processed by passage through a reverse osmosis membrane (R.O. water), chlorine added as a bacteriostat, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
other: Reverse osmosis deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Solutions of the test substance at concentrations of 1.4, 7 and 35 mg/mL were prepared once weekly at the Testing Facility and stirred continuously for at least 24 hours prior to dosage administration and stored at room temperature. During preparation of dosing solutions a correction factor was used to account for the high water content of the test substance.

VEHICLE
- Amount of vehicle: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the dosing formulations were conducted using gas chromatography and samples were taken three times from the top, middle and bottom of each dosing formulation prior to dosing and analyzed for verification of dose level concentration. All study samples analyzed for concentration were within the acceptance criteria of ±10% of their target values, with the exception of the Group 2 samples prepared on 28 December 2009 and on 04 and 11 January 2010 (mean recoveries of 123, 114 and 111%, respectively). Analysis of back-up samples confirmed that Group 2 samples prepared on 28 December 2009, 04 and 11 January 2010 were out of specification. For homogeneity, the relative standard deviation of the grand mean for all locations was ≤ 5% for all groups.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: maximum of 5 days
- Proof of pregnancy: vaginal plug referred to as Day 0 of pregnancy
Duration of treatment / exposure:
Day 6 - 18 of gestation
Frequency of treatment:
daily, 7 days/week
Dose / conc.:
7 mg/kg bw/day (actual dose received)
Dose / conc.:
35 mg/kg bw/day (actual dose received)
Dose / conc.:
175 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on adverse results in previous developmental toxicity studies of other perfluoroalkyl acids (PFAA), doses of 7, 35 and 175 mg/kg bw/day were selected for this study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: prior to dose administration and between one and two hours after dose administration and once each day during the post-dose period for clinical observations, abortions, premature deliveries and deaths; Mice were evaluated for adverse clinical signs observed during parturition and general condition of dam and litter during the postpartum period.
DETAILED CLINICAL OBSERVATION:
- Time scedule: Maternal behavior was evaluated on DLs 0, 4, 7, 14 and 20.

BODY WEIGHT: Yes
- Time schedule for examinations: on GD 0, and daily during the dosage and postdosage periods

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day: 20 postpartum
- Organs examined: gross pathology; liver (of 5 mice per group), uterus (number and distribution of implantation sites)

OTHER:
Mice that did not deliver a litter were sacrificed on GD 23 and dams with no surviving pups were sacrificed after the last pup was found dead or missing, presumed cannibalized. In these animals gross pathology was performed, the number and distribution of implantation sites were recorded after staining with 10% ammonium sulfide and livers were excised, weighed and frozen on dry ice. Duration of gestation (GD 0 to the day the first pup was observed) was evaluated.
Mice that died before scheduled termination were examined for the cause of death after the observation was made. In these animals gross pathology was performed. The lungs, trachea and esophagus were perfused and saved for possible future evaluation. The heart, kidneys, stomach and spleen were retained for possible histological evaluation. Gravid uterine weights were recorded. Pregnancy status and uterine contents of female mice were recorded. Conceptuses in utero were examined to the extent possible, using the same methods described for term fetuses/pups. The livers were excised, weighed and frozen on dry ice.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: Yes: all per litter; Necropsy of the pups included a single cut at the suture of the frontal and parietal bones of the skull, and the crosssectioned brain was examined for hydrocephaly.
- Other: litter sizes (all pups delivered) and pup viability at birth, number of offspring per litter (live and dead pups); Pups that died before initial examination were evaluated for vital status at birth by immersion of lungs in water.
Statistics:
Clinical observations and other proportional data were analysed using the Variance Test for Homogeneity of the Binomial Distribution. Continuous (parametric) data, such as body weights, organ weights, percentage of litter reaching a developmental landmark and percent mortality per litter were analysed as follows: Bartlett’s Test of Homogeneity of Variances was used to estimate the probability that the dosage groups had different variances. A non-significant result (p>0.001) indicated that an assumption of homogeneity of variance was not inappropriate, and the data were compared using the Analysis of Variance. If that test was significant (p≤0.05), the groups given the test substance were compared with the control group using Dunnett’s Test. If Bartlett’s Test was significant (p>0.001), the Analysis of Variance Test was inappropriate, and the data were analysed as non-parametric data, as follows: When 75% or fewer of the scores were tied, the Kruskal-Wallis Test was used to analyse the data, and in the event of a significant result (p≤0.05), Dunn’s Method of Multiple Comparisons was used to compare the groups given the test substance with the control group. When more than 75% of the scores were tied, Fisher’s Exact Test was used to compare the proportion of ties in the dosage group. Variables with graded count scores, such as litter size were analysed using Kruskal-Wallis Test (≤ 75%ies) as the non-parametric data. If this test was significant (p≤0.05), the groups given the test substance were compared with the control group using Dunn’s Test.
Indices:
The following indices were evaluated: fertility index (percentage of matings that result in pregnancies), gestation index (percentage of pregnancies that result in birth of live litters), viability indices (percentage of pups born that survive 4 and 7 days) and lactation index (percentage of pups born that survive 20 days)
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical observations included a red perivaginal substance in 1/20 mouse of 35 and 175 mg/kg bw/day group, respectively, and urine-stained abdominal fur in 1/20 mouse of 35 mg/kg bw/day group, respectively. All clinical observations during the gestation and lactation periods were considered unrelated to the test substance because: 1) the incidences were not dosage dependent; and 2) the observations occurred in only one mouse in a group.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
1/20 mouse of 7 mg/kg bw/day dosage group was sacrificed on Day 17 of gestation (DG 17) when it delivered its litter; and 1/20 mouse in the 35 mg/kg bw/day dosage group was sacrificed on Day 2 of lactation (DL 2) due to no surviving pups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
All body weight values were comparable among the four dosage groups and did not differ significantly.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The absolute weights of the liver and the ratio of the liver weight to the terminal body weight did not differ significantly between the groups.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Necropsy observations included numerous clear cysts in the liver (one 7 mg/kg bw/day dosage group mouse), clear fluid filled cyst in the capsule of the kidney (one 35 mg/kg bw/day dosage group mouse), thick walls of the uterus (one 7 mg/kg bw/day dosage group mouse) and clear fluid filled cysts in the uterus (one 35 mg/kg bw/day dosage group mouse). All necropsy observations were considered unrelated to the test substance administration because: 1) the incidences were not dosage dependent; or 2) the observations occurred in only one mouse.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Averages for implantation sites per delivered litter were comparable between the control and the treatment groups.
Total litter losses by resorption:
not specified
Early or late resorptions:
not examined
Dead fetuses:
not examined
Changes in pregnancy duration:
effects observed, non-treatment-related
Description (incidence and severity):
Durations of gestation were comparable between the control and treatment groups. One mouse in the 7 mg/kg bw/day dosage group delivered its litter before sceduled delivery at Day 17 of gestation (DG 17).
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Pregnancy occurred in 20, 17, 20 and 20 of the 20 mated female mice in the 0 (Vehicle), 7, 35 and 175 mg/kg/day dose groups, respectively.
Other effects:
no effects observed
Description (incidence and severity):
The number of dams delivering litter and the gestation index (number of dams with one or more liveborn pups/number of pregnant mice) were comparable between control and treatment groups.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
175 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed up to the highest dose tested.
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
175 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse effcts observed up to the highest dose tested.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The average pup weight per litter was significantly reduced on Day 1 postpartum in the 175 mg/kg bw/day dose group compared with the control
(see Table 2). However, lower pup weights were only noted on day 1 post partum. On Days 4, 7, 14 and 20 pup weights were similar to those of the control. The noted lower pups weights on Day 1 post partum were therefore considered to be not adverse.

This conclusion is also supported by the Iwai (2019) publication included in the reference list.

Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
The number of stillborn pups was statistically significantly increased (total n=3; 1.2%; mean 0.2 ± 0.7; p≤0.05) in the 175 mg/kg bw/day group compared with the control (n=0).

Supplementary information from publication (Iwai 2019):
When control groups from both study phases (Hoberman 2001 a and b) are pooled, the overall incidence of stillbirths of the combined control groups (n=4) represent 0.9% of pups delivered, compared with n=3 stillbirths representing 1.2% of pups delivered for the 175 mg/kg bw/d group. This difference is not statistically significant (p<0.05, Fisher exact test). Furthermore, the incidence of n=4 (1.2%) stillborn pups is within the range of the laboratories historical control data (0.4 - 1.4%). Accordingly, the incidence of stillborn pups is considered not to be test item-related.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter sizes were comparable among the four dosage groups and did not significantly differ.
Changes in postnatal survival:
effects observed, non-treatment-related
Description (incidence and severity):
With the exception of Day 1 post partum there was no difference in pup survival throughout the preweaning period. Day 4 and 7 viability indices as well as the lactation index at all dosage were similar to the control.
The number of live pups at 7 mg/kg bw/day was statistically significantly reduced at weaning. As this finding was not dose-dependent it was considered to be unrelated to treatment with the test item.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In two litters in the 175 mg/kg bw/day group, one pup each had corneal opacity and one pup had microphthalmia, respectively. One different litter in this dosage group also had one pup with a lenticular opacity.

Supplementary information from publication (Iwai 2019):
The historical control data for CD-1 mice, indicates that for any given study, there is greater than a 1 in 10 chance that an eye anomaly such as corneal opacity and microphthalmia will occur spontaneously in at least 1 pup in the absence of exposure to test material. The observations in this study are restricted to a single treatment group (175 mg/kg bw/day), and the overall incidence rate of 0.8% for both corneal opacity and microphthalmia is consistent with low reference ranges reported in the literature and historical control data reports for Crl: CD-1 mice. Accordingly, the ocular anomalities observed in this study are considered not to be test item-related.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
The day of eye opening did not differ among the groups.

No necropsy observations in the F1 generation pups were attributed to administration of the test substance at 175 mg/kg bw/day. 1/3 mice of 7 mg/kg bw/day group found dead, had a gas filled intestine; all other mice sacrificed on Day 20 postpartum appeared normal.

Key result
Dose descriptor:
NOAEL
Remarks:
development
Effect level:
175 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to and including 175 mg/kg bw/day.
Remarks on result:
other: NOAEL refers to F1 generation mice/pups.
Key result
Abnormalities:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related abnormal findings observed.
Key result
Developmental effects observed:
no

Results of F1 generation mice (postweaning period):

None of the effects observed in the pups preweaning persisted into the postweaning period. All F1 generation male and female mice survived to scheduled sacrifice. The gross pathology examination showed incidental changes in two animals: 1/20 male mice in the 175 mg/kg bw/day group had a clear fluid filled cyst in the liver and 1/20 female mice in the 35 mg/kg bw/day group had a dark flat red mass in the mesentery. Body weights and body weight gains of the F1 generation male and female mice were unaffected by maternal doses of the test substance up to 175 mg/kg bw/day. Maternal administration of the test substance did not affect the liver weights or its ratio to the terminal body weight. Sexual maturation was unaffected by maternal administration of the test substance. The average day on which preputial separation or vaginal patency occurred was comparable between the control and treatment groups.

Table 1: Adverse effects in litters and F1 pups

Dosage (mg/kg bw/day) a)

 

0 (vehicle)

7

35

175

Delivered a litter with one or more liveborn pubs

n

20

17

19

20

Pups delivered (total)

n

249

213

232

241

mean ± SD

12.4 ± 2.5

12.5 ± 3.0

12.2 ± 1.7

12.0 ± 2.1

Liveborn

mean ± SD (%)

12.4 ± 2.5

12.4 ± 3.4

12.2 ± 1.7

11.9 ± 2.5

n (%)

249 (100.0)

211 (99.1)

232 (100.0)

238 (98.8)

Stillborn

mean ± SD (%)

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

0.2 ± 0.7

n (%)

0 ( 0.0)

0 (0.0)

0 (0.0)

3 (1.2)**

Unknown vital status

n

0

2

0

0

Pups found dead or presumed cannibalised

 

 

 

 

 

Day 0

n/n (%)

0/249 (0.0)

0/211 (0.0)

0/232 (0.0)

4/238 (1.7)**

Days 1 - 4

n/n (%)

3/249 (1.2)

6/211 (2.8)

2/232 (0.9)

3/230 (1.3)

Days 5 - 7

n/n (%)

1/246 (0.4)

0/205 (0.0)

0/230 (0.0)

3/231 (1.3)

Days 8 - 14

n/n (%)

0/245 (0.0)

0/205 (0.0)

0/230 (0.0)

0/228 (0.0)

Days 15 - 20

n/n (%)

0/245 (0.0)

0/205( 0.0)

0/230 (0.0)

1/228 (0.4)

Day 4 viability index b)

%

98.8

97.2

99.1

97.0

n/n

246/249

205/211

230/232

231/238

Day 7 viability index c)

%

98.4

97.2

99.1

95.8

n/n

245/249

205/211

230/232

228/238

Lactation index d)

%

99.6

97.2

100

98.3

n/n

245/246

205/211**

230/230

227/231

Day(s) = postpartum Day(s)

a)    Dosage occurred on days 6 to 18 of gestation.

b)   Number of live pups on Day 4 postpartum/number of liveborn pups on Day 0 postpartum.

c)    Number of live pups on Day 7 postpartum/number of liveborn pups on Day 0 postpartum.

d)   Number of live pups on Day 20 postpartum/number of liveborn pups on Day 0 postpartum.

** significantly different from the vehicle control group value (p≤0.01).

Table 2. Pup weight/ litter (gram)

Dosage (mg/kg bw/day) a) 0 (vehicle) 7 35 175
Day 0  mean ± SD 1.6 ± 0.1 1.6 ± 0.1 1.6 ± 0.1 1.4 ± 0.2*
Day 4 mean ± SD 2.8 ± 0.3 2.8 ± 0.3 3.0 ± 0.3 2.7 ± 0.5
Day 7 mean ± SD 4.2 ± 0.6 4.2 ± 0.4 4.4 ± 0.4 4.2 ± 0.6
Day 14 mean ± SD 6.8 ± 1.2 6.7 ± 0.6 7.0 ± 0.7 6.8 ± 0.9
        [ 16]b          
Day 20 mean ± SD 10.2 ± 1.8 10.0 ± 1.2 10.8 ± 1.3 10.4 ± 1.4

Day = Day post-partum

[ ] = number of values averaged

a. Dosage occurred on days 6 through 18 of gestation.

b. Excludes values for litter 430, which had no surviving pups on day 2 postpartum.

* Significantly different from the vehicle control group value (p≤0.05).

 

Conclusions:
Based on the results of this study, the NOAEL for maternal systemic toxicity and fertility is 175 mg/kg bw/day. The NOAEL of the F1 generation for developmental toxicity is 175 mg/kg bw/day.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
29 Sep - 30 Dec 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Maternal treatment was only from day 6 to 18 post coitum.
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: In this combined developmental and perinatal/postnatal reproduction study toxic effects/disturbances resulting from test substance treatment of Crl:CD1(ICR) pregnant female mice and development of the embryo and fetus consequent to exposure of the dam from implantation to closure of the hard palate and during lactation was evaluated. This study was designed to evaluate the ICH Harmonised Tripartite Guideline stages C through F of the reproductive process and detect effects on gestation, parturition, lactation and maternal behavior in female mice, and on the development of the offspring of the treated female mice. Because manifestations of effects induced during this period may be delayed in the offspring, observations were continued through sexual maturity of the F1 generation mice.
- Short description of test conditions: Eighty presumed pregnant Crl:CD1(ICR) mice were randomly assigned to four dosage groups, 20 mice per group. Solutions of the test substance and/or the vehicle were administered orally once daily from Day 6 of gestation (GD 6) through DG 18. After completion of the 20 day postpartum period (PPD 20), F0 generation female mice were sacrificed and liver samples were collected from 5 mice per group for pharmacokinetic analysis; mice that did not deliver a litter were sacrificed on GD 23. Additionally, on PPD 20, all pups not selected for continued evaluation were sacrificed. F1 generation mice selected for continued evaluation were sacrificed on PPD 41. Blood and liver samples were collected from five mice per sex per group for pharmacokinetic analysis.
- Parameters analysed / observed: The following parameters were evaluated for F0 generation female mice: viability, clinical observations, body weights, body weight changes, maternal behavior, litter observations, natural delivery, pup body weights, dam and pup necropsy observations. The following parameters were evaluated for F1 generation male and female mice: viability, clinical observations, body weights, body weight changes, eye opening, age of sexual maturity and necropsy observations.
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
other: Crl:CD1(ICR)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Kingston, NY, USA
- Age at study initiation: approx. 61 days at day of arrival
- Weight at study initiation: 26.1 - 30.0 g (range)
- Housing: F0 was housed individually in stainless steel, wire bottom cages, except during the cohabitation and postpartum periods. During cohabitation, each pair of male and female mice was housed in the male mouse’s cage. Each dam and delivered litter was housed in a common nesting box with nesting material (Bed-o´cobs bedding) during the postpartum period.; F1 was housed paired in nesting boxes until PND 27, afterwards individually
- Diet (e.g. ad libitum): Certified Rodent Diet #5002 (PMI Nutrition International, Inc., St. Louis, MO, USA), ad libitum
- Water (e.g. ad libitum): Local water processed by passage through a reverse osmosis membrane (R.O. water), chlorine added as a bacteriostat, ad libutum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: Reverse osmosis deionised water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Solutions of the test substance at concentrations of 20, 70 and 100 mg/mL were prepared once weekly at the Testing Facility and stirred continuously for at least 24 hours prior to dosage administration and stored at room temperature. During preparation of dosing solutions a correction factor was used to account for the high water content of the test substance.

VEHICLE
- Amount of vehicle: 5mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the dosing formulations were conducted using gas chromatography and samples were taken three times from the top, middle and bottom of each dosing formulation prior to dosing and analyzed for verification of dose level concentration. All study samples analyzed for concentration were within the mean acceptance criteria of ±10% of their target values. For homogeneity, the relative standard deviation of the grand mean for all locations was ≤ 5% for all groups.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: maximum of 5 days
- Proof of pregnancy: vaginal plug referred to as Day 0 of pregnancy
Duration of treatment / exposure:
Day 6 - 18 of gestation
Frequency of treatment:
daily, 7 days/week
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
350 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previous combined developmental and perinatal/postnatal reproduction toxicity study (Hoberman, 2011a), mice were administered to the test substance at doses of 7, 35 and 175 mg/kg bw/day on DGs 6 through 18. No mortality related to the test substance occurred, and no adverse clinical signs occurred during this study. Due to a lack of observed systemic toxicity, dosages of 100, 350 and 500 mg/kg bw/day were selected for this study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: prior to dose administration and between one and two hours after dose administration and once each day during the post-dose period for clinical observations, abortions, premature deliveries and deaths; Mice were evaluated for adverse clinical signs observed during parturition and general condition of dam and litter during the postpartum period.
DETAILED CLINICAL OBSERVATION:
- Time scedule: Maternal behavior was evaluated on DLs 0, 4, 7, 14 and 20.

BODY WEIGHT: Yes
- Time schedule for examinations: on GD 0, and daily during the dosage and postdosage periods

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day: 20 postpartum
- Organs examined: gross pathology; liver (of 5 mice per group), uterus (number and distribution of implantation sites)

OTHER:
Mice that did not deliver a litter were sacrificed on GD 23 and dams with no surviving pups were sacrificed after the last pup was found dead or missing, presumed cannibalized. In these animals gross pathology was performed, the number and distribution of implantation sites were recorded after staining with 10% ammonium sulfide and livers were excised, weighed and frozen on dry ice. Duration of gestation (GD 0 to the day the first pup was observed) was evaluated.
Mice that died before scheduled termination were examined for the cause of death after the observation was made. In these animals gross pathology was performed. The lungs, trachea and esophagus were perfused and saved for possible future evaluation. The heart, kidneys, stomach and spleen were retained for possible histological evaluation. Gravid uterine weights were recorded. Pregnancy status and uterine contents of female mice were recorded. Conceptuses in utero were examined to the extent possible, using the same methods described for term fetuses/pups. The livers were excised, weighed and frozen on dry ice.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: Yes: all per litter; Necropsy of the pups included a single cut at the suture of the frontal and parietal bones of the skull, and the crosssectioned brain was examined for hydrocephaly.
- Other: litter sizes (all pups delivered) and pup viability at birth, number of offspring per litter (live and dead pups); Pups that died before initial examination were evaluated for vital status at birth by immersion of lungs in water.
Statistics:
Clinical observations and other proportional data were analysed using the Variance Test for Homogeneity of the Binomial Distribution. Continuous (parametric) data, such as body weights, organ weights, percentage of litter reaching a developmental landmark and percent mortality per litter were analysed as follows: Bartlett’s Test of Homogeneity of Variances was used to estimate the probability that the dosage groups had different variances. A non-significant result (p>0.001) indicated that an assumption of homogeneity of variance was not inappropriate, and the data were compared using the Analysis of Variance. If that test was significant (p≤0.05), the groups given the test substance were compared with the control group using Dunnett’s Test. If Bartlett’s Test was significant (p>0.001), the Analysis of Variance Test was inappropriate, and the data were analysed as non-parametric data, as follows: When 75% or fewer of the scores were tied, the Kruskal-Wallis Test was used to analyse the data, and in the event of a significant result (p≤0.05), Dunn’s Method of Multiple Comparisons was used to compare the groups given the test substance with the control group. When more than 75% of the scores were tied, Fisher’s Exact Test was used to compare the proportion of ties in the dosage group. Variables with graded count scores, such as litter size were analysed using Kruskal-Wallis Test (≤ 75%ies) as the non-parametric data. If this test was significant (p≤0.05), the groups given the test substance were compared with the control group using Dunn’s Test.
Indices:
The following indices were evaluated: fertility index (percentage of matings that result in pregnancies), gestation index (percentage of pregnancies that result in birth of live litters), viability indices (percentage of pups born that survive 4 and 7 days) and lactation index (percentage of pups born that survive 20 days)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Slight (p≤0.05) excess salivation in 3/20 mice in the 350 mg/kg bw/day group and slight to moderate (p≤0.01) salivation in 6/20 mice 500 mg/kg bw/day group.
All other clinical observations during the gestation and lactation periods were considered unrelated to the test substance because the incidences were not dosage dependent and the observations occurred in only one or two mice in a group. These clinical observations included slight/mild dehydration, dyspnea, tachypnea, decreased motor activity, ptosis, a red or dried red perivaginal substance, scant feces, hyperpnea, gasping and rales.
A purple area on the abdomen occurred in 10/20, 12/20, 13/20 and 9/20 mice in control, 100, 350 and 500 mg/kg bw/day groups, respectively. Abdominal distention occurred in 3/20 mice in the control and 100 mg/kg bw/day groups. These observations were considered not toxicologically relevant since they were secondary to the stress of nursing as the sign occurred late in lactation.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Totals of 3/20, 6/20, 1/20 and 3/20 dams were found dead in the control, 100, 350 and 500 mg/kg bw/day groups, respectively. Single deaths that occurred in the mid-dose and high-dose groups during the gestation period appear to have been related to administration of the test substance based on the timing of the deaths (GDs 8 and 13). The deaths in the control and low-dose group all occurred between days 13 and 16 of lactation. These deaths and possibly two of the three deaths that occurred in the highest dosage group appeared to be due to the stress of nursing. This is known to occur in mice.

Totals of 1, 0, 2 and 6 mice in the control, 100, 350 and 500 mg/kg bw/day groups, respectively, were sacrificed due to no surviving pups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gains during lactation were significantly reduced for Day 0 to 4 of lactation in the 350 and 500 mg/kg bw/day groups compared with the control. Although no additional significant differences occurred among the groups for body weight gain during lactation, the average gain during the entire lactation period was reduced in the 500 mg/kg bw/day group compared with the control. Body weight gains from Day 0 to 20 of lactation were 97.7%, 110.3% and 64.4% of the control group value.
Reduction in weight gain in 500 mg/kg bw/day group during lactation was considered to be toxicologically relevant.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Terminal body weights were comparable between the control and treatment groups. The absolute weights of the liver and the ratio of the liver weight to the terminal body weight did not differ significantly between the control and treatment groups.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Tan areas in the liver occurred in 1/20 mice in the 350 mg/kg bw/day group and 5/20 (p≤0.01) mice in the 500 mg/kg bw/day group. 1/20 mice each in the control and 100 mg/kg bw/day dosage group had a bent sternum proximal to the xiphoid process and 1/20 mice in the 500 mg/kg bw/day group had intestines that were distended with gas. Observations in the liver were considered to be presumably pathologic and therefore toxicologically relevant.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
The number of mice with stillborn pups and the number of mice with all pups dying on PPDs 0 to 3 were significantly increased (p≤0.01) in the 500 mg/kg/day dosage group compared to the control group values.The number of pups dying on Day 0 post partum was increased in the 350 mg/kg/day dosage group and significantly increased (p≤0.01) in the 500 mg/kg/day dosage group compared to the control group.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Pregnancy occurred in 19, 19, 20 and 18 of the 20 mated female mice in the control,100, 350 and 500 mg/kg bw/day groups, respectively.
Other effects:
no effects observed
Description (incidence and severity):
The number of dams delivering litter and the gestation index (number of dams with one or more liveborn pups/number of pregnant mice) were comparable between control and treatment groups.
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
350 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
gross pathology
mortality
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed at 100 mg/kg bw/day
Dose descriptor:
LOAEL
Remarks:
fertility
Effect level:
350 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: increased incidence of stillborn pups and pups dying on day 1 post partum
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed at 100 mg/kg bw/day.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
not examined
Description (incidence and severity):
Pup body weights in all treated groups were generally lower in the treated groups compared with the control group. Pup body weights were significantly reduced (p≤0.05 to p≤0.01) on postpartum Day 0 in the 100 mg/kg bw/day and higher dose groups compared with the control. Statistically significantly reduced pup body weights persisted in the 350 mg/kg bw/day group through postpartum Day 7 and in the 500 mg/kg bw/day group through postpartum Day 4. On postpartum Day 20 average pup weights per litter were 89%, 80% and 88% of the control group value for 100, 350 and 500 mg/kg bw/day groups, respectively. The lack of dose-dependency can be attributed to the differences in litter size among the groups.
The transiently reduced pup weights on Day 0 post partum at 100 mg/kg bw/day were considered not adverse as during the subsequent weighings no statistical significant difference compared to the control group was noted. This is supported by the fact that the examined developmental landmark (eye opening) was not delayed at 100 mg/kg bw/day.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
The number of dams with all pups dying on postpartum Day 0 to 3 were significantly increased (p≤0.01) in the 500 mg/kg bw/day dosage group compared with the control (5 dams with total litter loss compared with one dam in the control).
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
The average litter size in the 500 mg/kg bw/day group was significantly reduced at birth compared with the control. The average litter size was reduced throughout the lactation period with a significant reduction (p≤0.05) on postpartum Day (PPD) 4.
Changes in postnatal survival:
effects observed, treatment-related
Description (incidence and severity):
The number of pups dying on PPD 0 was increased in the 350 mg/kg bw/day dosage group and significantly increased (p≤0.01) in the 500 mg/kg bw/day group compared with the control. The number of pups dying on postpartum Days (PPDs) 1 to 4 in the 350 and 500 mg/kg bw/day dosage groups was significantly increased (p≤0.01) compared with the control.
The PPD 4 and 7 viability indices were significantly reduced (p≤0.01) compared with the control. The Day 7 viability index was significantly reduced (p≤0.05) in the 350 mg/kg bw/day group. The average number of surviving pups per litters was significantly reduced (p≤0.01) in the 500 mg/kg bw/day group for PPDs 4, 7, 14 and 20 compared with the control.
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
no effects observed
Description (incidence and severity):
All pups that survived to PPD 20, and were not continued on study appeared normal at necropsy.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The few clinical observations included scab, dehydration, tip of tail red or missing, not nursing, not nesting and ungroomed coat. These were considered to be incidental.
The average Day that 50% of the pups had open eyes was significantly later (p≤0.01) in the 350 mg/kg/day group compared with the control group value. The lack of a significant difference in the 500 mg/kg bw/day group may be related to the higher number of early pups deaths and reduced litter size in this group. The percentage of pups per litter with open eyes was significantly reduced (p≤0.05 to p≤0.01) in the 350 and 500 mg/kg bw/day groups on postpartum Day 14 compared with the control group value. The delays in eye opening are considered to result from delayed development (reduced body weights) and are therefore considered to be toxicologically relevant.
11/32 pups in the 500 mg/kg bw/day group that were found dead or stillborn had no milk in the stomach. This indicates, that pups were probably to weak to suckle or dams had no milk.
Dose descriptor:
LOAEL
Remarks:
development
Effect level:
350 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
Key result
Dose descriptor:
NOAEL
Remarks:
development
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse feects noted at 100 mg/kg bw/day.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
350 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Results of F1 generation mice (postweaning period):

Except of one male mouse in the 350 mg/kg bw/day group, which was found dead on PPD 23 with low body weight, normal appearance at necropsy and without clinical signs noted, all F1 generation male and female mice survived to scheduled sacrifice. All clinical observations in the F1 generation male and female mice were considered unrelated to maternal administration of the test substance because: 1) the incidences were not dosage dependent; 2) the observation occurred in only one mouse; and/or 3) the observation is common in this species and strain. These clinical observations were limited to common findings in the tail including constricted, bent, missing or purple. All F1 generation male mice appeared normal at necropsy. 1/20 females in the 100 mg/kg bw/day group had a small left kidney. Body weights of males were significantly reduced (p≤0.05) on PPD 21 in the 100 and 350 mg/kg bw/day groups and body weight gains were increased (p≤0.05 to p≤0.01) in the 100 and 350 mg/kg bw/day dosage group on PPDs 28 to 35 compared with the control.

In females, body weights were significantly reduced (p≤0.05 to p≤0.01) on PPDs 21 and 28 in the 100 and 350 mg/kg bw/day dosage group and on PPDs 35 and 41 (350 mg/kg bw/day only); and body weights were significantly reduced (p≤0.05) in the 500 mg/kg bw/day group on PPD 35 compared with the control. Body weight gains of females did not significantly differ between control and treatment groups for the postweaning period.

The ratio of the liver weight to the terminal body weight was significantly reduced (p≤0.05) in males in the 500 mg/kg bw/day group compared with the control. In females, liver weights or the ratio of liver weights to the terminal body weight were not affected.

Sexual maturation was unaffected by maternal dosages of the test substance as high as 175 mg/kg bw/day. The average day on which preputial separation or vaginal patency occurred was comparable among the four dosage groups.

Levels of test substance in liver homogenates

Levels PFH Ammonium salt in the livers from dams administered the 100 mg/kg/day dosage were all below the lower limit of quantization (0.02 μg/mL). In the 350 mg/kg bw/day dosage group, three of eight samples had analytical results that were quantifiable. The highest level (87.5 ug/mL) occurred in a mouse that was found dead on DG 13. The other mice had much lower levels of PFH Ammonium salt, but it is interesting that both of these mice were sacrificed early after their litters had died off. These were the only mice in this group that lost their litters. In the 500 mg/kg bw/day dosage group five of 16 samples had analytical results that were quantifiable. The highest level (98.4 ug/mL) occurred in a mouse that was found dead on DG 6. The other mice had much lower levels of PFH but each of these mice had litters that died early. Two samples that were below the lower limit of quantization were from mice that lost their litters. Based on these results, the variability in the quantization of PFH ammonium salt in the liver may be due to the time of sampling post the start of dosing. No detectable level of PFH was found in the liver homogenate from any F1 generation male or female pup.

Table 1. Maternal (F0) adverse effects

 

Dosage (mg/kg bw/ day)

Observations

0

100

350

500

dams found dead during lactation period

3/20

6/20

1/20

3/20

Body weight gain [g] on Day 0 - 4 of lactation

5.7 ± 2.1 (n=19) a

5.4 ± 1.8 (n=19)

3.8 ± 2.6 * (n=19) a)

2.8 ± 2.0 ** (n=17) a)

Dams with stillborn pubs n (%)

2/19 (10.5)

0/19 (0.0)

5/19 b) (26.3)

7/17 b) (41.2)**

Dams with all pups dying on Day 0 - 3 postpartum
n (%)

1/19 (5.3)

0/19 (0.0)

2/19 b) (10.5)

5/17 b) (31.3)**

a) Excludes values for mice that were sacrificed due to no surviving pups or found dead                            

b) Excludes mice that were found dead before delivery                                                             

* Significantly different from the control group value (p≤0.05)                                               

** Significantly different from the control group value (p≤0.01)        

Table 2. Live litter size

Dosage (mg/kg bw/day) a)   0 (vehicle) 100 350 500
Day 0  mean ± SD 11.4 ± 4.5 13.2 ± 1.6 12.0 ± 3.5 9.9 ± 2.9
          [ 13]b 
Day 4 mean ± SD 11.9 ± 3.8 13.0 ± 1.7 12.0 ± 3.6 9.9 ± 2.0*
    [ 18]b   [ 17]b [ 11]b 
Day 7 mean ± SD 11.9 ± 3.8 12.9 ± 1.6  11.8 ± 3.6 9.9 ± 2.0
    [ 18]b   [ 17]b  [ 11]b
Day 14 mean ± SD 11.9 ± 3.8 12.4 ± 1.4 11.6 ± 3.4  9.9 ± 2.0
    [ 18]b [ 15]c [ 17]b  [ 11]b 
Day 20 mean ± SD 11.9 ± 3.8 12.3 ± 1.2 11.6 ± 3.4 9.9 ± 2.0
    [ 18]b [ 15]c [ 17]b [ 11]b

Day = Day postpartum

[ ] = Number of valued averaged

a. Dosage occurred on days 6 through 18 of gestation.

b. Excludes values for litters that had no surviving pups.

c. Excludes litters with mortality of pups that remained on study after dam was found dead.

* Significantly different from the control group value (p≤0.05).

** Significantly different from the control group value (p≤0.01).                   

Table 3. Adverse effects in litters and F1 pubs

 

 Maternal dosage (mg/kg bw/ day)

Observations

0

50

100

200

pups delivered (total)

221

250

245

177

liveborn n (%)

217 (98.2)

250 (100.0)

232 (94.7)

150 (84.7)**

stillborn n (%)

4 (1.8)

0 (0.0)

5 (2.0)

16 (9.0)

unknown vital status a) n (%)

0

0

8

11

pups found dead or presumed cannibalised n/n (%)

 

Day 0

0/217 (0.0)

0/250 (0.0)

3/232 (1.3)

21/150 (14.0)**

Day 1 - 4

2/217 (0.9)

3/250 (1.2)

25/229 (10.9)**

20/129 (15.5)**

Day 4 viability index b) % (n/n)

99.1 (215/217)

98.8 (247/250)

87.9 (204/232)

72.7** (109/150)

Day 7 viability index c)

98.6 (214/217)

98.4 (246/250)

86.6* (201/232)

72.7** (109/150)

a) Maternal cannibalization or autolysis precluded identification of vital status at birth.

b) Number of live pups on day 4 postpartum/number of liveborn pups on day 0 postpartum.

c) Number of live pups on day 7 postpartum/number of liveborn pups on day 0 postpartum.

* Significantly different from the control group value (p≤0.05)

** Significantly different from the control group value (p≤0.01)        

Table 4. Body weights of F1 generation mice (postweaning period)

 

male body weight (g)

 

maternal dosage (mg/kg bw/day)

 

0

100

350

500

postpartum Day

 

 

 

 

21

12.5 ± 2.7

10.6 ± 2.2*

10.3 ± 2.8*

10.8 ± 2.4

28

22.8 ± 3.4

20.6 ± 3.4

20.6 ± 3.8

20.9 ± 3.7

35

29.0 ± 2.7

27.9 ± 2.8

28.0 ± 2.6

27.6 ± 2.7

41

32.1 ± 3.0

31.3 ± 2.6

31.0 ± 2.5

30.9 ± 2.7

 

female body weight (g)

 

maternal dosage (mg/kg bw/day)

 

0

100

350

500

postpartum Day

 

 

 

 

21

11.8 ± 2.4

10.1 ± 2.2*

9.8 ± 1.6**

10.8 ± 1.8

28

19.5 ± 2.4

17.7 ± 2.6*

16.4 ± 3.1**

18.1 ± 2.3

35

24.2 ± 1.8

23.2 ± 2.5

21.7 ± 2.8**

22.4 ± 1.5*

41

25.6 ± 2.0

24.7 ± 1.9

23.6 ± 2.0**

24.6 ± 1.7

* Significantly different from the control group value (p≤0.05)

** Significantly different from the control group value (p≤0.01)             

             

                    

Conclusions:
The maternal NOAEL for systemic toxicity of the test substance was 100 mg/kg bw/day based on single mortalities, reduced body weight gains during the lactation period and tan areas in the liver at 350 mg/kg bw/day. The NOAEL for fertility was 100 mg/kg bw/day based on an increased number of stillborn pups at 350 mg/kg bw/day and above.
For developmental toxicity a NOAEL of 100 mg/kg bw/day was considered based on decreased pup weights, reductions in viability indices, and delays in physical development in F1 generation mice from 350 mg/kg bw/day onwards.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available studies on postnatal development comprise adequate and reliable studies and are thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.
The available information on prenatal development comprises an adequate and reliable study from a reference substance with similar structure and intrinsic properties. Read-across is justified based on common precursors and breakdown products of hydrolysis and consistent trends in environmental fate, ecotoxicological and toxicological profile (refer to endpoint discussion for further details). The selected to study is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.
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

Justification for read-across

There are two studies available on the developmental toxicity of ammonium undecafluorohexanoate. The assessment of developmental/reproductive toxicity was additionally based on a study conducted with a suitable source substance as part of a read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

CAS 21615-47-4 (target substance)

Ammonium undecafluorohexanoate was tested in a combined developmental and perinatal/postnatal reproductive toxicity study (no guideline followed) in compliance with GLP (Hoberman, 2011a). The results of the study were also published in a paper by Iwai and Hoberman in 2014. Eighty presumed pregnant Crl:CD1(ICR) mice were randomly assigned to four dose groups, 20 mice per group. Solutions of the test substance at concentrations of 7, 35 and 175 mg/kg bw/day and/or the vehicle (reverse osmosis deionized water) were administered orally once daily from Day 6 of gestation (GD 6) through GD 18. The dosage volume was 5 mL/kg bw. The mice were allowed to litter and were observed until postnatal day 20. After completion of the 20-day postpartum period (PPD 20), F0 generation female mice were sacrificed. Mice that did not deliver a litter were sacrificed on GD 23. Additionally, on PPD 20, all pups not selected for continued (post-weaning) evaluation were sacrificed. F1 generation mice selected for continued evaluation were sacrificed on PPD 41.

The F0 dams were observed for mortality and clinical signs daily, and the body weights were recorded daily. During necropsy on PPD 20 a gross pathology examination was performed, and the liver and uterus was examined. The number of implantations was recorded. All F0 mice survived until scheduled sacrifice, with the exception of one mouse in the 7 mg/kg bw/day group that was sacrificed on GD 17 when it delivered its litter before scheduled delivery; and one mouse in the 35 mg/kg bw/day group that was sacrificed on PPD 2 due to no surviving pups. These occurrences were considered to be incidental. No substance-related effects on clinical parameters or fertility parameters were found. Body weight and body weight gains during gestation and lactation and liver weights were unaffected by the treatment. No changes were observed during necropsy. Pregnancy occurred in 20, 17, 20 and 20 of the 20 mated female mice in the control, 7, 35 and 175 mg/kg bw/day groups, respectively. All pregnant dams delivered a litter.

The number of stillborn pups and pups dying on PPD 1 was significantly increased. The average pup weight per litter was transiently, although significantly reduced on PPD 1 in the 175 mg/kg bw/day dosage group compared with the control. The lower pup weights were only noted on day 1 post partum. On Days 4, 7, 14 and 20 pup weights were similar to those of the control. The noted lower pups weights on Day 1 post partum were therefore considered not to be an adverse effect. Two litters in the 175 mg/kg bw/day group had a pup each with corneal opacity and one pup each with microphthalmia. One different litter in this dosage group also had 1/2 pups with a lenticular opacity. The day of eye opening did not differ among the groups. No necropsy observations in the F1 generation pups were attributed to administration of the test substance at 175 mg/kg bw/day.

All F1 generation male and female mice (post-weaning period) survived to scheduled sacrifice and showed no substance-related effects on body weight and liver weight. Incidental necropsy observations in the F1 generation female and male mice occurred in one mouse each in the 35 and 175 mg/kg bw/day dosage groups, respectively. One male mouse had a clear fluid filled cyst in the liver and one female mouse had a dark flat red mass in the mesentery. Sexual maturation was unaffected by maternal exposure to the test substance.

The results of this study were re-assessed in a publication by Iwai et al. (2019). This publication presents a supplemental data analysis and evaluation of the findings from the original study. The original study has been cited as supporting a lowest-observed-adverse-effect level of 175 mg/kg bw/day and a no observed-adverse-effect level of 35 mg/kg bw/day for developmental effects. Although the in-study statistical analysis was accurate in terms of quantifying statistical significance, given the low incidence of findings, the purpose of the publication was to extend the analysis and interpretation of findings by pooling the control group information from both phases of the same study (entered in IUCLID as Hoberman 2011 a and b), comparing the study findings to the incidence rates for stillbirths, postpartum viability for this species and strain of mouse observed for similar studies conducted by the same laboratory, and evaluating data on the incidence and range of spontaneous eye abnormalities reported in the literature. Based on this supplemental evaluation, the findings of increased incidence of stillborn pups was considered not to be treatment-related because when control groups from both study phases (Hoberman 2001 a and b) were pooled, the overall incidence of stillbirths of the combined control groups (n=4) was 0.9% of pups delivered, compared with n=3 stillbirths representing 1.2% of pups delivered for the 175 mg/kg bw/day group. This difference is not statistically significant (p<0.05, Fisher exact test). Furthermore, the incidence of n=4 (1.2%) stillborn pups fell within the range of the laboratory’s historical control data (0.4 - 1.4%).

Furthermore the paper concluded that the incidence of ocular effects in the study is most likely unrelated to treatment with the test item. This conclusion is supported by the historical control data for CD-1 mice, which demonstrates that for any given study there is greater than a 1 in 10 chance that an eye anomaly such as corneal opacity and microphthalmia will occur spontaneously in at least 1 pup in the absence of exposure to a test substance. The observations in this study are restricted to a single treatment group (175 mg/kg bw/day), and the overall incidence rate of 0.8% for both corneal opacity and microphthalmia is consistent with low reference ranges reported in the literature and historical control data for Crl: CD-1 mice.

Based on the results of this study, the additional analyses and subsequent revised conclusion, the NOAEL for maternal systemic toxicity and fertility is 175 mg/kg bw/day and the NOAEL of F1 generation for developmental toxicity is 175 mg/kg bw/day.

CAS 21615-47-4 (target substance)

Ammonium undecafluorohexanoate was tested in a second combined developmental and perinatal/postnatal reproductive toxicity study (no guideline followed) in compliance with GLP (Hoberman, 2011b). The study was conducted according to the same study protocol as the first study (Hoberman, 2011a), using higher dose levels of 100, 350 and 500 mg/kg bw/day.

3/20, 6/20, 1/20 and 3/20 dams were found dead in the control, 100, 350 and 500 mg/kg bw/day groups, respectively. Single deaths that occurred in the mid- and high- dose groups during the gestation period appear to have been related to administration of the test substance based on the timing of the deaths (GD 8 and 13) other death appeared to be caused by stress of nursing. 1/20, 0/20, 2/20 and 6/20 mice in the control, 100, 350 and 500 mg/kg bw/day groups, respectively, were sacrificed due to no surviving pups. Clinical observations during the gestation period considered related to the test substance was slight to moderate excess salivation in the 350 and 500 mg/kg bw/day group. Body weight gains during lactation were significantly reduced for postpartum Day (PPD) 0 to 4 in the 350 and 500 mg/kg bw/day groups compared with the control. Although no additional significant differences were noted among the control and treatment groups for body weight gain during the lactation period, the average gain during the entire lactation period was reduced in the 500 mg/kg bw/day group, compared with the control. During necropsy, tan areas in the liver were observed in 1/20 mice in the 350 mg/kg bw/day group and 5/20 (p≤0.01) mice in the 500 mg/kg bw/day group. The absolute weights of the liver and the ratio of the liver weight to the terminal body weight did not differ significantly between the control and treatment groups.

The number of mice with stillborn pups and the number of mice for which all the pups died on PPD 0 to 3 was significantly increased in the 500 mg/kg bw/day group compared with the control. The pregnancy duration, the number of dams delivering litter and the gestation index were comparable between control and treatment groups.

The number of pups dying on Days 1 to 4 in the 350 and 500 mg/kg bw/day groups was significantly increased compared with the control. The number of pups dying on PPD 0 was increased (non-significantly) in the 350 mg/kg bw/day group and significantly increased in the 500 mg/kg bw/day group compared with the control group.

No clinical observations in the F1 generation pups were attributed to the treatment. Eye opening occurred significantly later in the 350 and 500 mg/kg bw/day groups. Pup body weights were transiently but statistically significantly reduced on postpartum Day 0 in the 100 mg/kg bw/day and higher dose groups compared with the control. Significantly reduced pup body weights were observed in the 350 mg/kg bw/day group through PPD 7 and in the 500 mg/kg bw/day group through PPD 4 but were comparable with control until terminal body weight. The transiently reduced pup weights on Day 0 post partum at 100 mg/kg bw/day were considered not adverse as during the subsequent body weight measurements no statistically significant difference was noted, compared with the control group. This is supported by the observation that the developmental landmark (eye opening) was not delayed at 100 mg/kg bw/day.

All pups that survived to PPD 20, and were not selected for post-weaning evaluations appeared normal at necropsy.

In F1 generation mice selected for post-weaning evaluations, one male mouse in the 350 mg/kg bw/day group was found dead on PPD 23. The male had low body weight, normal appearance at necropsy and no clinical signs were noted prior to the death. All clinical observations in the F1 generation mice were considered to be unrelated to maternal treatment with the test substance and animals appeared normal at necropsy. Body weights of males were significantly reduced (p≤0.05) on PPD 21 in the 100 and 350 mg/kg bw/day groups and body weight gains were increased (p≤0.05 to p≤0.01) in the 100 and 350 mg/kg bw/day group on PPDs 28 to 35 compared with the control. In females, body weights were significantly reduced (p≤0.05 to p≤0.01) on PPD 21 and 28 in the 100 and 350 mg/kg bw/day group and on PPD 35 and 41 (350 mg/kg bw/day only); and body weights were significantly reduced (p≤0.05) in the 500 mg/kg/day group on PPD 35 compared with the control. The ratio of the liver weight to the terminal body weight was significantly reduced in the 500 mg/kg bw/day group of male mice compared to the control group value, but was unaffected in females. Sexual maturation was unaffected by maternal dosages of the test substance as high as 500 mg/kg bw/day.

The maternal NOAEL for systemic toxicity of the test substance was 100 mg/kg bw/day based on increased mortality, reduced body weight gains during the lactation period and tan areas in the liver in 5/20 animals at 350 mg/kg bw/day and above. The NOAEL for fertility was 350 mg/kg bw/day based on an increased number of stillborn pups at 500 mg/kg bw/day.

For developmental toxicity a NOAEL of 100 mg/kg bw/day was considered based on decreased pup weights at 350 mg/kg bw/day and above. Reduced viability and delays in physical development in F1 generation mice were observed in the 350 and 500 mg/kg bw/day groups.

CAS 2923-26-4

Sodium perfluorohexanoate (CAS 2923-26-4) was tested in a prenatal developmental toxicity study according to OECD guideline 414 and in compliance with GLP (Chemours, 2007). Groups of 22 time-mated Crl:CD(SD) rats were administered formulations of the test substance in water by once daily gavage on gestation days (GD) 6-20 at daily dose levels of 20, 100, and 500 mg/kg bw/day in water at 5 mL/kg bw. Control rats were administered the vehicle (water) only. During the in-life portion of the study, The dams were observed for mortality and clinical signs twice daily, and the body weights were recorded daily during GD 6-21. On GD 21, all dams were euthanized and a gross external and visceral examination was performed. The uterus of each pregnant female was removed and the uterine contents were examined and described; all fetuses were removed and individually identified, weighed, sexed, and examined for external alterations. Approximately one-half of the fetuses were examined for soft tissue alterations; all fetuses were examined for skeletal alterations.

There were no test substance-related deaths or gross postmortem findings in dams at any treatment level. In the high-dose group, overall weight gain (GD 6-21) and overall net weight gain (GD 6-21 weight gain excluding the gravid uterus weight) were 19% and 26% lower than controls, respectively. In addition, daily mean body weights on GD 19-21 and net body weight on GD 21 were 5-7% lower than controls. Beginning on GD 12, mean daily food consumption at 500 mg/kg bw/day was 10%-12% lower than controls for most of the subsequent intervals. Although these decreases in daily food consumption resulted in only a small decrease in overall food consumption (5% lower than controls on GD 6- 21), the effect on food consumption was considered adverse at this dosage since it was of sufficient magnitude to have an adverse impact on body weight gain during late gestation. Test substance-related clinical signs of toxicity occurred only in one severely affected animal in the 500 mg/kg bw/day group, and consisted of nasal discharge, lung noise, and wet/stained skin/fur, which occurred concomitantly with periods of body weight loss and markedly decreased food consumption in this animal. Although limited to one animal and transient, these clinical signs were considered test substance-related. There were no test substance-related gross postmortem findings at any dose level.

Developmental toxicity was limited to a decrease (10% lower than controls) in fetal weight at 500 mg/kg bw/day. There were no test substance-related fetal external, visceral or skeletal malformations or variations observed at any dose level.

Under the conditions of this study, the NOAEL for maternal systemic toxicity and developmental toxicity was 100 mg/kg bw/day, based on reductions in maternal body weight parameters and food consumption, and decreased fetal weight at 500 mg/kg bw/day. Since reduced pup body weights are assumed to be a secondary effect of maternal systemic toxicity at the same dose level of 500 mg/kg bw/day, sodium perfluorohexanoate is therefore concluded not to present a developmental hazard.

Conclusion on developmental toxicity

For the target substance ammonium undecafluorohexanoate (CAS 21615-47-4) two studies are available on developmental toxicity performed according to the ICH Harmonised Tripartite Guidelines stages C-F pointing to developmental toxicity in the presence of maternal toxicity only (Hoberman, 2011 a and b, with additional evaluations in Iwai, 2019). The guidelines used are different from the OECD 414 guideline, which is recommended under the REACH Regulation (EC) No. 1907/2006. Important differences are: the study was performed in mice (rat is the preferred rodent species in OECD guideline 414), mice littered and offspring were sacrificed on post-partum Day 20 or 41 (OECD guideline 414: animals are sacrificed on gestation Day 20-21, before littering, so that the fetuses can be assessed for malformations). The studies are considered relevant for classification as they provide valuable information on the postnatal development of F1 offspring, on the potential effects in an additional species (the mouse) and as the studies are performed with the target substance, ammonium undecafluorohexanoate .

In addition to data on the target substance, a study with the source substance sodium perfluorohexanoate (CAS 2923-26-4) performed according to OECD guideline 414 in rats was available. In this study, developmental toxicity was observed only at dose levels also causing maternal toxicity, and the effects are therefore considered to be secondary to the maternal toxicity (Chemours, 2007).

As explained in the Analogue justification (see IUCLID section 13) similar toxicological properties are assumed for the target and the source substance and were evident in the observed effects on the dams and related effects on pups considered to be secondary to maternal toxicity. As the studies performed with the target substance were not performed according to the requirements laid down in the REACH Regulation (EC) No. 1907/2006, Annex VIII, 8.7, and because the study performed with the source substance resulted in a lower NOAEL, the study conducted with the source substance was selected as the overall key study and its results were used for hazard assessment. Therefore, based on the read-across approach the NOAEL for developmental toxicity is considered to be 100 mg/kg bw/day for the target substance ammonium undecafluorohexanoate (CAS 21615-47-4).

Justification for classification or non-classification

Based on the available studies with ammonium undecafluorohexanoate and the analogue read-across approach, the available data on toxicity to reproduction does not meet the classification criteria according to Regulation (EC) 1272/2008 and is therefore conclusive but not sufficient for classification.

Additional information