Ammonium undecafluorohexanoate

Developmental toxicity / teratogenicity

Administrative data

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.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Reference 2

Reference Type:

publication

Title:

Oral (Gavage) Combined Developmental and Perinatal/Postnatal Reproduction Toxicity Study of Ammonium Salt of Perfluorinated Hexanoic Acid in Mice

Author:

Iwai, H. and Hoberman, A.M.

Year:

2014

Bibliographic source:

Int J Toxicol 33(3): 219-237

Reference 3

Reference Type:

publication

Title:

Addendum to Iwai and Hoberman (2014) - Reassessment of Developmental Toxicity of PFHxA in Mice

Author:

Iwai, H., Hoberman, A.M., Goodrum, P.E., Mendelsohn, E., Anderson, J.K.

Year:

2019

Bibliographic source:

Int J Toxicol

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium undecafluorohexanoate

EC Number:

244-479-6

EC Name:

Ammonium undecafluorohexanoate

Cas Number:

21615-47-4

Molecular formula:

C6H4F11NO2

IUPAC Name:

2,2,3,3,4,4,5,5,6,6,6-Undecafluorohexanoic acid, ammonium salt (1:1)

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

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.

Examinations

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)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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.

Effect levels (maternal animals)

1

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.

2

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.

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

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.

Effect levels (fetuses)

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.

Fetal abnormalities

Key result

Abnormalities:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related abnormal findings observed.

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

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).

 

Applicant's summary and conclusion

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.