Perfluamine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

PTPA

IUPAC Name:

PTPA

Details on test material:

- Name of test material (as cited in study report): (CF3CF2CF2)3N, PTPA, 338-83-0

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3M Company, Batch 3435912
- Expiration date of the lot/batch: 07 July, 2021
- Purity test date: 12 December, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING : None, dosed neat.

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: 11 to 15 weeks old
- Weight at study initiation: between 194 and 266 g
- Fasting period before study: None
- Housing: On arrival and following randomization females were housed individually in Macrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS -
J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 43-54
- Air changes (per hr): At least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 March, 2019 To: 11 April, 2019

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test article was dosed neat.

VEHICLE: None

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The purity of the test article was determined via NMR spectormeter using a Bruker Avance 300 digital NMR spectrometer equipped with Bruker 5 mm BBFO 300 MHz Z-gradient high resolution-ATM Probe

Details on mating procedure:

Time-mated female Wistar Han Rats were received from Charles River Laboratories Deutschland, Sulzfeld, Germany. The females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating).

Duration of treatment / exposure:

The test item was administered to the animals of Groups 2, 3 and 4 at the appropriate dose volumes (and Elix Water to animals of Group 1) once daily by oral gavage 7 days a week from Day 6 to Day 20 post-coitum, inclusive.

Frequency of treatment:

Daily

Duration of test:

Day 6 to Day 20 post-coitum, inclusive.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1: Elix water control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

22 females per dose

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: The doses were based on the results of an OECD 421 study conducted on the test article.
- Rationale for animal assignment (if not random): Random

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed
from the cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were performed at least once daily, beginning on Day 2 post-coitum and
lasting up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION: Yes
Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 21 post-coitum
- Organs examined: number of corpora lutea, organ weights ((gravid) uterus and thyroid gland), uterine contents

Other: The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (T3, T4, TSH), gross necropsy findings, number of corpora lutea, organ weights ((gravid) uterus and thyroid gland), uterine contents and histopathologic examination (thyroid gland).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.

Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

The following pairwise comparisons were made:
Group 2 vs Group 1
Group 3 vs Group 1
Group 4 vs Group 1

Datasets with at least 3 groups were compared using Dunnett-test.

Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test. Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant. No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

Number of corpora lutea, the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, ano-genital distance.

Historical control data:

Results were compared to historical Charles River data.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No adverse clinical signs were noted during the observation period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights, body weight gain and weight gain corrected for gravid uterus of treated females remained in the same range as controls over the treatment period.
After correction for uterus weight, the body weight of one female at necropsy (No. 80, at 1000 mg/kg/day) appeared to be slightly lower (less than 2%) when compared to its body weight on Day 6 post-coitum. Since a large variation in body weight gain between individual animals was also observed in other groups, this single finding was considered of no toxicological relevance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight was similar to the control level over the study period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum levels of TSH, total T3 and T4 were considered to be unaffected by treatment up to 1000 mg/kg/day.

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):

Thyroid weights and thyroid to body weight ratios of treated animals were considered to be similar to those of control animals.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations

Other effects:

no effects observed

Description (incidence and severity):

The numbers of pregnant females, corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

The numbers of pregnant females, corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.

Description (incidence and severity):

The number of pre- and post-implantation losses in control and test groups were similar and in the range of normal biological variation.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Number of resorptions was unaffected by treatment.

Early or late resorptions:

no effects observed

Description (incidence and severity):

Number of early and late resorptions was unaffected by treatment.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on litter size of any group.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Pregnancy duration was unaffected by treatment.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The number of pregnant femals was not impacted by treatment.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical biochemistry

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

food efficiency

gross pathology

histopathology: neoplastic

histopathology: non-neoplastic

maternal abnormalities

mortality

necropsy findings

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no toxicologically relevant effects on fetal body weights (both sexes) noted by treatment up to 1000 mg/kg/day.

The statistically significant differences apparent for the mean male fetal weights in Groups 3 and 4 were considered the result of slightly low mean fetal weights in the males of the control group, in comparison with the historical control data, rather than being indicative of a relation to treatment.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on litter size of any group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to 1000 mg/kg/day.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on litter size of any group.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on litter size of any group.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on fetal external morphology following treatment up to 1000 mg/kg/day.

Two externally malformed fetuses were observed in this study and in both cases, the lower jaw was affected. Fetus A058-02 at 300 mg/kg/day had a small lower jaw and in control fetus A011-03 the lower jaw was absent. Skeletal examination substantiated these findings, whereby also other skull bones appeared to be affected. The single occurrence and group distribution of these jaw findings did not indicate a test item relationship and were therefore considered spontaneous in origin. External variations were not seen in any group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on skeletal morphology following treatment up to 1000 mg/kg/day.

Aside from the underlying malformations of the two fetuses with jaw findings (A011-03 and A058-02 of the control and 300 mg/kg/day group, respectively), four different malformations were revealed skeletally in this study. Bent limb bones occurred at 100 mg/kg/day (fetus A039-01), 300 mg/kg/day (fetus A062-02 and A064-10) and 1000 mg/kg/day (fetus A072-10) and was the only malformation that occurred in all test item treated groups. Other malformations noted in treated groups were costal cartilage anomaly at 1000 mg/kg/day (fetus A067-05) and vertebral centra anomaly at 300 mg/kg/day (fetus A058-02). Besides a vertebral anomaly in control fetus A011-01 that also had a costal cartilage anomaly, no other malformations occurred. The single occurrence and group distribution of these malformations did not indicate a dose relationship and all were seen previously in historical controls, therefore they were considered chance findings. Among skeletal variations, two findings showed a remarkable group distribution. Bent ribs occurred at a mean incidence of 9.0%, 21.1%, 11.6% and 30.5% per litter in the control, 100, 300 and 1000 mg/kg/day groups, respectively. The incidences at 100 and 1000 mg/kg/day were statistically significantly increased compared to the control value and the high dose value was even outside the historical control data range (0.8 – 27.4% per litter). However, it is known that bent ribs represent a reversible finding and since no dose relationship could be established, these increases were considered not treatment-related. The other notable variation was the finding of 14 rudimentary ribs, which occurred at an incidence of 41.8%, 66.8%, 61.4% and 51.3% per litter in the control, 100, 300 and 1000 mg/kg/day groups, respectively. The 100 mg/kg/day value was statistically significantly increased compared to the control value, but lower than the historical control maximum value of 72.0% per litter. Therefore, and due to absence of a dose-relationship, this was considered to have occurred by chance. The other variations that were noted occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within the range of available historical control data. Therefore, they were not considered treatment-related.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on visceral morphology following treatment up to 1000 mg/kg/day.

Visceral malformations occurred in two fetuses of the control group and in one fetus at 1000 mg/kg/day. The fetus at the high dose, A075-04, had a right-sided aortic arch which was also observed in control fetus A014-01 and was therefore considered spontaneous in origin.
The visceral malformation in control fetus A011-03 comprised situs inversus in combination with absence of the lower jaw (see external malformations above).
In addition, three types of visceral variations (small supernumerary liver lobes, absent accessory lung lobe and dilated ureter) were observed among several fetuses in this study. Based on the low incidence of occurrence, their distribution over the dose groups and the fact that the incidence of each finding remained within the range of historical control data, they were considered not to be related to treatment.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

external malformations

skeletal malformations

visceral malformations

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Body Weights (Grams) Summary Parental Generation	Corrected for gravid uterus	0 mg/kg	100 mg/kg	300 mg/kg	1000 mg/kg
Post Coitum
Day 2	Mean	214	217	214	216
	S.D.	22.3	18.5	20.8	22.7
	N	22	22	22	21
Day 6	Mean	231	234	230	233
	S.D.	20.3	18.1	21.0	20.5
	N	22	22	22	21
Day 9	Mean	239	242	240	243
	S.D.	19.4	16.8	19.9	20.3
	N	22	22	22	21
Day 12	Mean	253	254	253	255
	S.D.	19.7	16.9	20.0	21.3
	N	22	22	22	21
Day 15	Mean	266	267	266	267
	S.D.	20.2	16.4	19.9	22.1
	N	22	22	22	21
Day 18	Mean	294	296	295	297
	S.D.	23.6	18.0	23.5	25.5
	N	22	22	22	21
Day 21	Mean	330	332	331	333
	S.D.	26.8	21.0	26.4	29.1
	N	22	22	22	21

Body Weights Gain (%) Summary - Parental Generation	Corrected for gravid uterus	0 mg/kg	100 mg/kg	300 mg/kg	1000 mg/kg
Post Coitum
Day 2	Mean	-7	-7	-7	-8
	S.D.	2.4	2.1	2.3	2.9
	N	22	22	22	21
Day 6	Mean	0	0	0	0
	S.D.	0.0	0.0	0.0	0.0
	N	22	22	22	21
Day 9	Mean	4	4	4	4
	S.D.	1.4	1.7	1.6	1.5
	N	22	22	22	21
Day 12	Mean	9	9	10	9
	S.D.	2.4	2.2	2.2	2.8
	N	22	22	22	21
Day 15	Mean	15	14	16	15
	S.D.	3.1	3.0	2.8	3.9
	N	22	22	22	21
Day 18	Mean	28	27	28	28
	S.D.	4.4	3.3	3.3	4.2
	N	22	22	22	21
Day 21	Mean	43	43	44	43
	S.D.	6.6	4.5	4.3	5.8
	N	22	22	22	21

Maternal Survival and Pregnancy Status	0 mg/kg	100 mg/kg	300 mg/kg	1000 mg/kg
# Females in Study	22	22	22	22
Females Examined at Scheduled Necropsy	22	22	22	22
Nongravid	0	0	0	1 (4.5%)
Gravid	22 (100%)	22 (100%)	22 (100%)	21 (95.5%)
Gravid with resorptions only	0	0	0	0
Gravid with viable fetuses	22 (100%)	22 (100%)	22 (100%)	21 (100%)
Total Females Gravid	22 (100%)	22 (100%)	22 (100%)	21 (95.5%)

Summary of Fetal Data at Necropsy		0 mg/kg	100 mg/kg	300 mg/kg	1000 mg/kg
Sex: Male	Total	134	111	122	116
	Mean	6.1	5.0	5.5	5.5
	S.D.	1.8	1.65	2.39	1.72
Sex: Female	Total	102	126	116	116
	Mean	4.6	5.7	5.3	5.5
	S.D.	1.5	1.93	2.68	1.81
Viable Fetuses	Total	236	237	238	232
	Mean	10.7	10.8	10.8	11.0
	S.D.	1.72	1.54	1.76	1.72
Dead Fetuses	Total	0	0	0	1
	Mean	0	0	0	0
	S.D.	0	0	0	0.22
Early Resorptions	Total	9	9	11	5
	Mean	0.4	0.4	0.5	0.2
	S.D.	0.59	0.73	0.96	0.62
Late Resorptions	Total	0	0	0	0
	Mean	0	0	0	0
	S.D.	0	0	0	0
Post Implanation Loss	Total	9	9	11	6
	Mean	0.4	0.4	0.5	0.3
	S.D.	0.59	0.73	0.96	0.64
Implantation Sites	Total	245	246	249	238
	Mean	11.1	11.2	11.3	11.3
	S.D.	1.36	1.37	1.36	1.74
Corpora Lutea	Total	256	261	256	247
	Mean	11.6	11.9	11.6	11.8
	S.D.	1.22	1.46	1.40	1.81
Pre Implantation Loss	Total	11	15	7	9
	Mean	0.5	0.7	0.3	0.4
	S.D.	0.96	0.89	0.57	0.51
Fetal Weights in Grams	Total	NA	NA	NA	NA
	Mean	5.1	5.1	5.2	5.2
	S.D.	0.28	0.26	0.27	0.27
Number of Gravid Females	Total	22	22	22	21
	Mean	-	-	-	-
	S.D.	-	-	-	-
None significantly different from control group

Summary of Fetal Data at Necropsy (% per litter)		0 mg/kg	100 mg/kg	300 mg/kg	1000 mg/kg
CORPORA LUTEA	Mean	11.6	11.9	11.6	11.8
	S.D.	1.22	1.46	1.40	1.81
	N	22	22	22	21
IMPLANTATION SITES	Mean	11.1	11.2	11.3	11.3
	S.D.	1.36	1.37	1.36	1.74
	N	22	22	22	21
VIABLE FETUSES (%)	Mean	96.0	96.3	95.4	97.6
	S.D.	5.84	6.63	8.63	5.44
	N	22	22	22	21
DEAD FETUSES (%)	Mean	0.0	0.0	0.0	0.5
	S.D.	0.0	0.0	0.0	2.18
	N	22	22	22	21
EARLY RESORPTIONS (%)	Mean	4	3.7	4.6	1.9
	S.D.	5.84	6.63	8.63	5.18
	N	22	22	22	21
LATE RESORPTIONS (%)	Mean	0.0	0.0	0.0	0.0
	S.D.	0.0	0.0	0.0	0.0
	N	22	22	22	21
TOTAL RESORPTIONS (%)	Mean	4.0	3.7	4.6	1.9
	S.D.	5.84	6.63	8.63	5.18
	N	22	22	22	21
PRE-IMPLANTATION LOSS (%)	Mean	4.1	5.5	2.6	3.5
	S.D.	7.78	7.37	4.60	4.25
	N	22	22	22	21
POST-IMPLANTATION LOSS (%)	Mean	4.0	3.7	4.6	2.4
	S.D.	5.84	6.63	8.63	5.44
	N	22	22	22	21
MALES (%)	Mean	565.5	47.2	52.0	50.1
	S.D.	12.53	14.36	21.75	14.46
	N	22	22	22	21
FEMALES (%)	Mean	43.5	52.8	48.0	49.9
	S.D.	12.53	14.36	21.75	14.46
	N	22	22	22	21
MALE FETAL WEIGHTS (g)	Mean	5.2	5.3	5.4*	5.4*
	S.D.	0.29	0.28	0.28	0.26
	N	22	22	22	21
FEMALE FETAL WEIGHTS (g)	Mean	4.9	5.0	5.1	5.1
	S.D.	0.28	0.24	0.26	0.28
	N	22	22	22	21
COMBINED FETAL WEIGHTS (g)	Mean	5.1	5.1	5.2	5.2
	S.D.	0.28	0.26	0.27	0.27
	N	22	22	22	21
MALE AGD (MM)	Mean	2.74	2.72	2.76	2.77
	S.D.	0.203	0.202	0.143	0.147
	N	22	22	22	21
FEMALE AGD (MM)	Mean	1.25	1.19	1.23	1.27
	S.D.	0.223	0.213	0.184	0.174
	N	22	22	22	21
CORRECTED MALE AGD	Mean	1.59	1.56	1.58	1.58
	S.D.	0.115	0.112	0.075	0.079
	N	22	22	22	21
CORRECTED FEMALE AGD	Mean	0.74	0.69	0.72	0.74
	S.D.	0.132	0.124	0.112	0.102
	N	22	22	22	21
FETAL WEIGHTS COMPARED USING DUNNETT'S TEST * = Significantly different from the control group at 0.05

Summary of Fetuses with Malformations (Absolute No.)	0 mg/kg	100 mg/kg	300 mg/kg	1000 mg/kg
Number Examined Externally	236	237	238	232
Lower Jaw – Absent or Small	1	0	1	0
Number Examined Viscerally	119	119	119	115
Aortic arch – right-sided	1	0	0	1
Situs Inversus	1	0	0	0
Number Examined Skeletally	118	118	119	117
Vertebral anomaly with or without associated rib anomaly	1	0	0	0
Costal cartilage anomaly	1	0	0	1
Bent limb bones	0	1	2	1
Vertebral centra anomaly	0	0	1	0
Total number with malformations
External:	1	0	1	0
Soft tissue:	2	0	0	1
Skeletal:	1	1	3	2
Combined:	3	1	3	3

Summary of Litter Proportions of Malformations - % Per Litter		0 mg/kg	100 mg/kg	300 mg/kg	1000 mg/kg
Number of litters examined externally		22	22	22	21
Lower Jaw – absent or small	Mean	0.5	0.0	0.4	0.0
	S.D.	2.37	0.00	1.78	0.00
Number of litters examined viscerally		22	22	22	21
Aortic Arch – Righ-sided	Mean	0.8	0.0	0.0	1.0
	S.D.	3.55	0.00	0.00	4.36
Situs Inversus	Mean	0.9	0.0	0.0	0.0
	S.D.	4.26	0.0	0.0	0.0
Number of litters examined skeletally		22	22	22	21
Vertebral anomaly with or without associated rib anomaly	Mean	0.9	0.0	0.0	0.0
	S.D.	4.26	0.0	0.0	0.0
Costal cartilage anomaly	Mean	0.9	0.0	0.0	0.8
	S.D.	4.26	0.0	0.0	3.64
Bent limb bones	Mean	0.0	0.9	1.8	1.0
	S.D.	0.0	4.26	5.88	4.36
Vertebral centra anomaly	Mean	0.0	0.0	0.8	0.0
	S.D.	0.0	0.0	3.55	0.0
Total Malformations
Percent/litter with external malformations	Mean	0.5	0.0	0.4	0.0
	S.D.	2.37	0.0	1.78	0.0
Percent/litter with soft tissue malformations	Mean	1.7	0.0	0.0	1.0
	S.D.	5.42	0.0	0.0	4.36
Percent/litter with skeletal malformations	Mean	0.9	0.9	2.6	1.7
	S.D.	4.26	4.26	6.66	5.54
Total percent/litter with malformations	Mean	2.2	0.9	2.2	2.7
	S.D.	7.37	4.26	6.03	6.80

Summary of Fetuses with Variations (Absolute No.)	0 mg/kg	100 mg/kg	300 mg/kg	1000 mg/kg
Number Examined Externally	236	237	238	232
Number with findings	0	0	0	0
Number Examined Viscerally	119	119	119	115
LIVER- SMALL SUPERNUMERARY LOBE(S)	5	1	2	0
LUNG- ABSENT ACCESSORY LOBE	1	0	0	0
URETER(S)- DILATED	0	1	0	0
Number Examined Skeletally	118	118	119	117
14TH RUDIMENTARY RIB(S	51	78	74	62
14TH FULL RIB(S)	9	12	8	13
STERNEBRA(E) MALALIGNED	9	12	8	13
7TH CERVICAL OSSIFICATION SITE(S)	3	7	5	5
BENT RIB(S)	11	24	13	34
REDUCED OSSIFICATION OF THE SKULL	16	13	13	15
PELVIC GIRDLE- CAUDAL SHIFT	10	9	7	12
METACARPAL(S) AND/OR METATARSAL(S) UNOSSIFIED	5	1	3	2
STERNUM- SUPERNUMERARY OSSIFICATION SITE	1	0	1	0
VERTEBRAL CENTRA- REDUCED OSSIFICATION	0	1	4	1
STERNEBRA(E) #5 AND/OR #6 UNOSSIFIED	0	0	1	0
7TH CERVICAL FULL RIB(S)	1	0	0	0
VERTEBRAL ARCHES- REDUCED OSSIFICATION	1	0	1	0
STERNEBRA(E)- BRANCHED	1	1	1	1
METACARPAL(S) AND/OR METATARSAL(S)- MALPOSITIONED	0	1	0	0

Summary of Fetuses with Variations (Absolute No.)		0 mg/kg	100 mg/kg	300 mg/kg	1000 mg/kg
Number Examined Externally		22	22	22	21
Number with findings		0	0	0	0
Number Examined Viscerally		22	22	22	21
LIVER- SMALL SUPERNUMERARY LOBE(S)	Mean	4.2	1.1	1.6	0.0
	S.D.	10.14	5.33	6.09	0.0
LUNG- ABSENT ACCESSORY LOBE	Mean	0.8	0.0	0.0	0.0
	S.D.	3.55	0.0	0.0	0.0
URETER(S)- DILATED	Mean	0.0	0.9	0.0	0.0
	S.D.	0.0	4.26	0.0	0.0
Number Examined Skeletally		22	22	22	21
14TH RUDIMENTARY RIB(S	Mean	41.8	66.8**	61.44	51.2
	S.D.	32.11	24.59	29.63	28.24
14TH FULL RIB(S)	Mean	8.0	9.7	6.4	11.7
	S.D.	18.13	14.18	12.89	22.35
STERNEBRA(E) MALALIGNED	Mean	11.3	8.3	10.0	8.6
	S.D.	12.87	9.41	10.84	12.62
7TH CERVICAL OSSIFICATION SITE(S)	Mean	2.2	6.4	3.9	4.3
	S.D.	7.27	12.58	8.63	9.59
BENT RIB(S)	Mean	9.0	21.1*	11.6	30.5**
	S.D.	18.96	27.09	14.65	32.05
REDUCED OSSIFICATION OF THE SKULL	Mean	13.8	11.0	10.9	13.5
	S.D.	24.72	15.33	20.76	20.66
PELVIC GIRDLE- CAUDAL SHIFT	Mean	9.4	7.4	6.0	11.0
	S.D.	19.50	16.78	10.47	21.43
METACARPAL(S) AND/OR METATARSAL(S) UNOSSIFIED	Mean	4.4	0.8	2.4	1.4
	S.D.	17.26	3.55	6.27	4.30
STERNUM- SUPERNUMERARY OSSIFICATION SITE	Mean	0.9	0.0	0.9	0.0
	S.D.	4.26	0.0	4.26	0.0
VERTEBRAL CENTRA- REDUCED OSSIFICATION	Mean	0.0	0.8	3.7	0.7
	S.D.	0.0	3.55	8.65	3.12
STERNEBRA(E) #5 AND/OR #6 UNOSSIFIED	Mean	0.0	0.0	0.8	0.0
	S.D.	0.0	0.0	3.55	0.0
7TH CERVICAL FULL RIB(S)	Mean	0.9	0.0	0.0	0.0
	S.D.	4.26	0.0	0.0	0.0
VERTEBRAL ARCHES- REDUCED OSSIFICATION	Mean	0.9	0.0	0.9	0.0
	S.D.	4.26	0.0	4.26	0.0
STERNEBRA(E)- BRANCHED	Mean	0.6	0.8	0.9	1.2
	S.D.	3.05	3.55	4.26	5.46
METACARPAL(S) AND/OR METATARSAL(S)- MALPOSITIONED	Mean	0.0	0.8	0.0	0.0
	S.D.	0.0	3.55	0.0	0.0
Total Variations
PERCENT/LITTER with External Variations	Mean	0.0	0.0	0.0	0.0
	S.D.	0.0	0.0	0.0	0.0
PERCENT/LITTER WITH SOFT TISSUE VARIATIONS	Mean	5.0	2.0	1.3	0.0
	S.D.	10.43	6.67	6.09	0.0
PERCENT/LITTER WITH SKELETAL VARIATIONS	Mean	64.5	86.5	80.0	78.6
	S.D.	30.9	21.2	19.77	23.64
TOTAL PERCENT/LITTER WITH VARIATIONS	Mean	69.5	88.5	81.3	78.6
	S.D.	28.85	22.10	18.61	23.64
* = Significantly different from the control group at 0.05 ** = Significantly different from the control group at 0.01

Clinical Biochemistry Summary -Parental Generation	End of Treatment	0 mg/kg	100 mg/kg	300 mg/kg	1000 mg/kg
TSH ulU/mL	Mean	0.357	0.316	0.334	0.396
	S.D.	0.220	0.166	0.282	0.278
	N	22	22	22	21
Total T3 ug/dL	Mean	59.4	55.1	61.3	55.7
	S.D.	8.4	9.4	10.1	8.8
	N	19	22	21	20
Total T4 ug/dL	Mean	2.58	2.4	2.32	2.55
	S.D.	0.71	0.59	0.37	0.58
	N	22	22	22	21

Organ Weights (Gram) Summary - Parental Generation	End of Treatment	0 mg/kg	100 mg/kg	300 mg/kg	1000 mg/kg
Body Wgt	Mean	330	332	331	333
	S.D.	27	21	26	29
	N	22	22	22	21
Thyroids	Mean	0.015	0.016	0.015	0.016
	S.D.	0.004	0.004	0.003	0.003
	N	22	22	22	21

Organ Weight Ratios(%) Summary – Parental Females	End of Treatment	0 mg/kg	100 mg/kg	300 mg/kg	1000 mg/kg
Body W	Mean	330	332	331	333
	S.D.	27	21	26	29
	N	22	22	22	21
Thyroids	Mean	0.005	0.005	0.005	0.005
	S.D.	0.001	0.001	0.001	0.001
	N	22	22	22	21

Historical Control Data Rat: Crl: WI(Han) (Outbred, SPF-quality)	Mean of Study Means	Total No. Animals in Control Group: 1097
Endpoint	Mean	S.D.	Median	Min	Max	P5	P95	Fetuses	Litters
Mean Male Body Weight (g)	5.4	0.11	5.4	5.0	5.5	5.2	5.5	-	-
Mean Female Body Weight (g)	5.1	0.12	5.1	4.7	5.3	4.9	5.3	-	-
Limb Bone(s) Bent (% Per Litter)1	0.3	0.71	0.0	0.0	2.3	@	@	5	3
14^thRudimentary Ribs (% per litter)	51.2	11.98	53.2	19.0	72.0	23.1	71.8	3033	970
Rib(s) – Bent (% per litter)	13.7	6.37	12.8	0.8	27.4	2.1	25.8	757	401
Liver – Small supernumerary Lobe(s) (% per litter)	4.6	2.8	4.8	0.0	9.6	0.0	9.1	266	221
Lung – Abnormal Lobation (% per litter)	0.0	0.11	0.0	0.0	0.8	0.0	0.0	1	1
Ureter(s) – Dialated (% per litter)	0.5	1.32	0.0	0.0	8.5	0.0	2.3	57	32
@ Insufficient number of data for calculation.1Based on 12 datasets.

Applicant's summary and conclusion

Conclusions:

Based on the results of the study, the prenatal developmental No Observed Adverse Effect Level (NOAEL) is 1000 mg/kg/day.

Executive summary:

The prenatal developmental toxicity potential of the test article was evaluated in female Wistar Han rats. The study was conducted according to OECD 414 in compliance with OECD GLP regulations. Time-mated female rats (22/dose) were exposed from Day 6 to Day 20 post-coitum, inclusive, via oral gavage to 0 (control), 100, 300, or 1000 mg/kg/day test article. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (T3, T4, TSH), gross necropsy findings, number of corpora lutea, organ weights ((gravid) uterus and thyroid gland), uterine contents and histopathologic examination (thyroid gland). In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, ano-genital distance, external, visceral and skeletal malformations and developmental variations. No maternal toxicity was observed in any dose group. No developmental toxicity was observed in any dose group. Based on the results of the study, the prenatal developmental No Observed Adverse Effect Level (NOAEL) is 1000 mg/kg/day.