Perfluamine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / 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 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

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
- Solubility and stability of the test substance in the solvent/vehicle: NA, dosed neat
TREATMENT OF TEST MATERIAL PRIOR TO TESTING : None, dosed neat.

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The strain is recommended per OECD 421.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 10-12 weeks
- Weight at study initiation: Males: 250-350 g, Females: 200-250 g
- Fasting period before study: None
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals will be group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). During the mating phase, males and females will be cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males will be housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females will be individually housed in Macrolon plastic cages (MIIItype, height 18 cm). During the lactation phase, females will be housed in Macrolon plastic cages (MIII type, height 18 cm). Pups will be housed with the dam. The cages will contain appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and will be equipped with water bottles. The housing conditions will be maintained unless deemed inappropriate by the Study Director and/or Clinical Veterinarian. The room(s) in which the animals will be kept will be documented in the study records. Animals will be separated during designated procedures/activities. Each cage will be clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) will be provided ad libitum throughout the study, except during designated procedures. The feed is analyzed by the supplier for nutritional components and environmental contaminants. Results
of the analysis are provided by the supplier and are on file at the Test Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum): Municipal tap water will be freely available to each animal via water bottles. Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. It is considered that there are no known contaminants in the water that would interfere with
the objectives of the study.
- Acclimation period: At least 5 days.

DETAILS OF FOOD AND WATER QUALITY: See diet and water sections above.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): At least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 02 January, 2019 To: 21 March, 2019

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS: The test item, MTDID 42916 was administered via oral gavage as received. An adequate amount of the test item was dispensed into daily aliquots, which were stored at room temperature.

- VEHICLE : None

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Maximum 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol: None

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

The test item was used as received from the Sponsor; therefore, samples for dose formulation analysis were not collected by the Test Facility.

Duration of treatment / exposure:

The test item (and Elix water for the control group) was administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days. Males were treated for 29 days, up to and during the mating period. Females that delivered were treated for 51-64 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 40-54 days.

Frequency of treatment:

Daily

Details on study schedule:

From the mating period onwards, the following parameters were recorded for each female:
male number paired with, mating date, confirmation of pregnancy and delivery day.

Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed.

Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Elix water control (Group 1)

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:

10

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: Other repeated dose studies.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: NA
- Post-exposure recovery period in satellite groups: NA

Positive control:

None

Examinations

Parental animals: Observations and 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 once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy. During the dosing period, these observations were performed directly after dosing. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. Thirteen days after start of mating
additional body weights were determined from females Nos. 59, 64 and 74 to check their pregnancy status because of the absence of evidence of mating. A terminal body weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION: Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum
and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Blood of F0-animals was collected on the day of scheduled necropsy. F0-males were fasted over night with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight. Blood of F1-animals was collected on PND 4 and PND 14-16, if possible. This
was performed in the necropsy room.
- Anaesthetic used for blood collection: Yes (Isofluorane)
- Animals fasted: Males were fasted, females were not
- How many animals:
All F0 animals and F1 animals at culling. On PND 14-16 separate blood samples were collected from two pups per litter (from one male and one female).
- Parameters checked: Serum T4 levels

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous.

Sperm parameters (parental animals):

Parameters examined in [F0] male parental generations:
testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes, on PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Pups were observed daily for general health/mortality. The number of live and dead pups was determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings. Pups showing pain, distress or discomfort which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail.Live pups were weighed individually on PND 1, 4, 7 and 13. Sex was externally determined for all pups on PND 1 and 4. Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight. All male pups in each litter were examined for the number of areola/nipples on PND 13.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.]

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: None

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: None

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes, All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A vet
erinary pathologist, or other suitably qualified person, was available. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any
former implantation sites and the number of corpora lutea was recorded in addition.

HISTOPATHOLOGY: Yes
The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
All animals: Gross lesions/masses
All animals of Groups 1-4: Epididymis, coagulation gland, prostate gland, seminal vesicle gland, thyroid gland, ovaries, testes

Postmortem examinations (offspring):

The following parameters were examined in F1 offspring:
Pups were observed daily for general health/mortality. The number of live and dead pups was determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings. Pups showing pain, distress or discomfort which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail.Live pups were weighed individually on PND 1, 4, 7 and 13. Sex was externally determined for all pups on PND 1 and 4. Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight. All male pups in each litter were examined for the number of areola/nipples on PND 13.

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.

Reproductive indices:

Mating index, precoital time, fertility index, gestation index, duration of gestation, lactation index

Offspring viability indices:

Post-implationation survival index, live birth index, percentatge life males at first litter check, percentage live females at first litter check, viability index.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One male (no.24, Group 3) showed abnormal gait during the last week of mating (fourth week of treatment). In the study files it was recorded that it did not properly used its hind legs. A few days before planned necropsy, veterinary inspection was conducted on this male. It was noted that it had its ears moved backwards and a rough fur, possible indications of pain. Moreover, a slight body weight loss was observed during the two weeks mating period. Furthermore, the male didn’t succeed in successful mating with its female partner. The cause of the injury of the hind legs was unknown, but might be related to the other findings in this male. It was considered an incidental finding that was not related to treatment. Alopecia noted in a single female (no. 56, Group 2) during the lactation phase occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study.
In the absence of a dose-related trend and at the incidence observed, these findings in a single male and female were considered to be incidental findings and of no toxicological significance.

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:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight gain were considered to have been unaffected by treatment. In high dose males, a slightly lower body weight gain was observed after treatment at 1000 mg/kg/day, achieving levels of statistical significance on several occasions when compared to controls. However, the average body weight gain in high dose animals was normal in comparison with the historical control data for rats of this strain and age, whereas that in control males was slightly higher than expected. Since the difference in mean body weights between control and high dose males did not exceed 3% during the study, no toxicological significance was attached to this finding. In high dose females at 1000 mg/kg/day, the mean body weights were slightly lower when compared to controls throughout the study, achieving a level of statistical significance on some occasions, i.e. Day 4 and 7 post coitum and Day 1 of lactation. However, during the study the relative difference in mean body weights varied from approximately 2% to 5% between controls and high dose females, indicating that the body weight gain was comparable between the two groups. Since the relative differences in mean body weights between controls and high dose females were similar at start and at the end of the study (i.e. approximately 3% lower), no toxicological significance was attached to this finding.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight in the high dose group at 1000 mg/kg/day (Group 4) was similar to the control group (Group 1) over the treatment period.

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:

no effects observed

Description (incidence and severity):

Serum levels of T4 in F0 males were considered not to be affected by treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

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.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No test article-related changes were observed in the testes, epididymis, or spermatogenic profiling.

Reproductive performance:

no effects observed

Description (incidence and severity):

No reproductive indices were impacted by treatment.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Litter size was considered not affected by treatment.
The mean number of living pups per litter at first litter check were 10.5 and 12.4 for the control and 1000 mg/kg/day groups, respectively.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

After similar mean body weights at birth (PND 1), a small difference became apparent in male and female pups between the control and high dose groups during the lactation period. On PND 13, approximately 10% lower mean body weights were observed in high dose male and female pups, achieving a level of statistical significance when compared to the concurrent controls. Since body weight gain in high dose pups was in the same range as the historical control data for pups of this strain and age, and that in the control pups was slightly higher, the difference in mean body weight between these groups was considered not treatment related and of no toxicological relevance. A relation with the slightly lower litter size in the control group is suspected.

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:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were slightly higher in the high dose group at 1000 mg/kg/day, not achieving levels of statistical significance when compared to controls and also remained within the range of historical control data for rat-pups of this age and strain.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.

Treatment at 1000 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.

Treatment at 1000 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related macroscopic findings were noted among pups at sacrifice.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.

Treatment at 1000 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

clinical biochemistry

gross pathology

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Male Body Weights (Grams) Summary
		Group 1 (Control)	Group 4 (1000 mg/kg)
Pre-Mating
Day 1	Mean	288	286
	Standard Deviation	7.9	8.1
	N	10	10
Day 8	Mean	319	312
	Standard Deviation	10.9	8.7
	N	10	10
Mating Period
Day 1	Mean	340	330
	Standard Deviation	13.5	10.1
	N	10	10
Day 8	Mean	352	343
	Standard Deviation	15.9	10
	N	10	10
Day 15	Mean	368	357
	Standard Deviation	16.1	8.9
	N	10	10
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Male Body Weight Gain (%) Summary
		Group 1 (Control)	Group 4 (1000 mg/kg)
Pre-Mating
Day 1	Mean	0	0
	Standard Deviation	0.0	0.0
	N	10	10
Day 8	Mean	11	9*
	Standard Deviation	1.9	1.7
	N	10	10
Mating Period
Day 1	Mean	18	16*
	Standard Deviation	3.2	1.4
	N	10	10
Day 8	Mean	22	20
	Standard Deviation	3.3	2.2
	N	10	10
Day 15	Mean	28	25*
	Standard Deviation	3.6	2.2
	N	10	10
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Historical Control Data – Male Body Weights (Grams)
Days	Mean	Std. Dev.	N	P5	P95
1	291	16.8	580	270.0	317.0
8	315	18.2	575	289.0	345.0
15	335	21.0	575	303.0	369.0
22	346	22.5	574	312.0	386.0
29	361	25.5	563	322.0	402.0

Historical Control Data – Male Body Weight Gain (%)
Days	Mean	Std. Dev.	N	P5	P95
1	0	0.0	580	0.0	0.0
8	8	2.5	575	4.0	12.4
15	15	3.9	575	8.8	21.4
22	19	4.9	574	11.7	27.2
29	24	6.2	563	13.8	33.8

Female Body Weights (Grams) Summary
		Group 1 (Control)	Group 4 (1000 mg/kg)
Pre-Mating
Day 1	Mean	225	218
	Standard Deviation	9.9	10.7
	N	10	10
Day 8	Mean	227	226
	Standard Deviation	9.4	9.8
	N	10	10
Mating Period
Day 1	Mean	236	230
	Standard Deviation	8.3	11.6
	N	10	10
Day 8	Mean		-
	Standard Deviation		-
	N	0 x	0 x
Day 15	Mean		-
	Standard Deviation		-
	N	0 x	0 x
Day 22	Mean		-
	Standard Deviation		-
	N	0 x	0 x
Post-Coitum
Day 0	Mean	237	228
	Standard Deviation	8.5	10.9
	N	8	9
Day 4	Mean	252	239*
	Standard Deviation	11.4	10.8
	N	8	9
Day 7	Mean	257	246*
	Standard Deviation	11.0	10.8
	N	8	9
Day 11	Mean	271	260
	Standard Deviation	12.8	11.5
	N	8	9
Day 14	Mean	281	271
	Standard Deviation	13.6	13.6
	N	8	9
Day 17	Mean	302	291
	Standard Deviation	14.8	14.8
	N	8	9
Day 20	Mean	340	327
	Standard Deviation	19.3	16.9
	N	8	9
Lactation
Day 1	Mean	267	252*
	Standard Deviation	15.0	13.4
	N	8	10
Day 4	Mean	279	270
	Standard Deviation	8.1	15.9
	N	8	10
Day 7	Mean	289	282
	Standard Deviation	13.3	12.6
	N	8	10
Day 13	Mean	304	294
	Standard Deviation	16.2	13.3
	N	8	10
X- Body weights excluded during mating period.
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Female Body Weight Gain (%) Summary
		Group 1 (Control)	Group 4 (1000 mg/kg)
Pre-Mating
Day 1	Mean	0	0
	Standard Deviation	0.0	0.0
	N	10	10
Day 8	Mean	1	3
	Standard Deviation	2.5	1.7
	N	10	10
Mating Period
Day 1	Mean	5	5
	Standard Deviation	2.6	2.3
	N	10	10
Day 8	Mean		-
	Standard Deviation		-
	N	0 x	0 x
Day 15	Mean		-
	Standard Deviation		-
	N	0 x	0 x
Day 22	Mean		-
	Standard Deviation		-
	N	0 x	0 x
Post-Coitum
Day 0	Mean	0	0
	Standard Deviation	0.0	0.0
	N	8	9
Day 4	Mean	7	5
	Standard Deviation	3.1	1.3
	N	8	9
Day 7	Mean	9	8
	Standard Deviation	3.4	1.7
	N	8	9
Day 11	Mean	14	14
	Standard Deviation	4.1	3.1
	N	8	9
Day 14	Mean	19	19
	Standard Deviation	4.1	3.5
	N	8	9
Day 17	Mean	28	28
	Standard Deviation	4.5	3.4
	N	8	9
Day 20	Mean	44	43
	Standard Deviation	6.5	4.8
	N	8	9
Lactation
Day 1	Mean	0	0
	Standard Deviation	0.0	0.0
	N	8	10
Day 4	Mean	5	7
	Standard Deviation	4.5	1.9
	N	8	10
Day 7	Mean	8	12*
	Standard Deviation	4.3	1.7
	N	8	10
Day 13	Mean	14	17
	Standard Deviation	3.7	3.8
	N	8	10
X- Body weights excluded during mating period.
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Historical Control Data – Female Body Weights (Grams)
Days	Mean	Std. Dev.	N	P5	P95
1	220	11.6	475	201.0	238.0
8	224	12.0	474	205.0	243.0
15	230	12.3	474	209.0	250.0
22	253	13.9	31	234.0	277.0
29	260	19.1	21	233.0	287.0

Historical Control Data – Female Body Weight Gain (%)
Days	Mean	Std. Dev.	N	P5	P95
1	0	0.0	475	0.0	0.0
8	2	2.9	474	-1.9	6.4
15	4	3.5	474	-0.8	10.1
22	12	3.9	31	6.2	17.9
29	15	4.9	21	9.8	23.6

Clinical Biochemistry Summary - Males
		Group 1 (Control)	Group 4 (1000 mg/kg)
End of Treatment
TSH	Mean	-	-
ulU/mL	Standard Deviation	-	-
	N	0	0
Total T4	Mean	4.22	4.75
ug/dL	Standard Deviation	0.96	0.90
	N	10	10

Macroscopic Findings Summary - Males
	Group 1 (Control)	Group 4 (1000 mg/kg)
End of Treatment
Animals Examined	10	10
Animals without findings	9	5
Animals affected	1	5
Epididymides (Nodule(s))	0	3
Coagulating Glands
Left side: Mishshapen	0	1
Agenesis	0	1
Thyroid Gland (Enlarged)	0	1
Iliac lymph Node (Englarged)	0	1
Eyes (Exophthalmus)	1	0

Macroscopic Findings Summary - Females
	Group 1 (Control)	Group 4 (1000 mg/kg)
End of Treatment
Animals Examined	10	10
Animals without findings	5	9
Animals Affected	5	1
Uterus
Contains fluid	2	0
Thyroid Gland
Discoloration	1	0
Adrenal Gland
Discoloration	0	1
Mandibular lymph node
Focus/foci	4	0

Organ Weights (Gram) Summary - Males
		Group 1 (Control)	Group 4 (1000 mg/kg)
End of Treatment
Body Weight	Mean	347	338
	Standard Deviation	16	8
	N	10	10
Liver	Mean	8.45	8.17
	Standard Deviation	0.46	0.35
	N	10	10
Thyroids	Mean	0.016	0.017
	Standard Deviation	0.003	0.004
	N	10	10
Testes	Mean	3.27	3.43
	Standard Deviation	0.28	0.23
	N	10	10
Prostate Gland	Mean	0.801	0.753
	Standard Deviation	0.094	0.108
	N	10	10
Epididymides	Mean	1.085	1.130
	Standard Deviation	0.108	0.070
	N	10	10
Seminal Vesicles	Mean	1.256	1.256
	Standard Deviation	0.202	0.229
	N	10	10

Organ/Body Weight Ratios (%) Summary - Males
		Group 1 (Control)	Group 4 (1000 mg/kg)
End of Treatment
Body Weight	Mean	347	338
(Gram)	Standard Deviation	16	8
	N	10	10
Liver (%)	Mean	2.44	2.42
	Standard Deviation	0.18	0.07
	N	10	10
Thyroids (%)	Mean	0.005	0.005
	Standard Deviation	0.001	0.001
	N	10	10
Testes (%)	Mean	0.94	1.02
	Standard Deviation	0.10	0.08
	N	10	10
Prostate Gland (%)	Mean	0.231	0.223
	Standard Deviation	0.032	0.032
	N	10	10
Epididymides (%)	Mean	0.312	0.334
	Standard Deviation	0.029	0.020
	N	10	10
Seminal Vesicles (%)	Mean	0.363	0.372
	Standard Deviation	0.066	0.067
	N	10	10

Organ Weights (Gram) Summary - Females
		Group 1 (Control)	Group 4 (1000 mg/kg)
End of Treatment
Body Weight	Mean	292	291
	Standard Deviation	30	16
	N	10	10
Liver	Mean	11.6	12.01
	Standard Deviation	2.48	1.17
	N	10	10
Thyroids	Mean	0.015	0.014
	Standard Deviation	0.003	0.003
	N	10	10
Ovaries	Mean	0.127	0.118
	Standard Deviation	0.031	0.018
	N	10	10

Organ/Body Weight Ratios (%) Summary- Females
		Group 1 (Control)	Group 4 (1000 mg/kg)
End of Treatment
Body Weight (grams)	Mean	292	291
	Standard Deviation	30	16
	N	10	10
Liver (%)	Mean	3.93	4.12
	Standard Deviation	0.50	0.32
	N	10	10
Thyroids (%)	Mean	0.005	0.005
	Standard Deviation	0.001	0.001
	N	10	10
Ovaries (%)	Mean	0.045	0.040
	Standard Deviation	0.017	0.006
	N	10	10

Reproduction Data Summary
	Group 1 (Control)	Group 4 (1000 mg/kg)
Females Paired	10	10
Females Mated	10	10
Pregnant Females	8	10
Females with living pups on day 1	8	10
Mating Index (%)	100	100
Fertility Index (%)	80	100
Gestation Index (%)	100	100

Precoital Time – F0 Post Coitum
Day of the Pairing Period	Group 1 (Control)	Group 4 (1000 mg/kg)
1	6	1
2	1	6
3	1	3
4	2	-
Median Precoital Time	1	2
Mean Precoital Time	1.9	2.2
N	10	10

Implantation Sites Summary
At Necropsy		Group 1 (Control)	Group 4 (1000 mg/kg)
Implantations	Mean	12.5	13.4
	Standard Deviation	3.7	2.3
	N	8	10

F0-Generation - Lactation
	Group 1 (Control)	Group 4 (1000 mg/kg)
Litters – Total	8	10
Duration of Gestation
Mean (+)	21.4	21.8
Standard Deviation	0.5	0.4
N	8	10
Dead Pups and First Litter Check
Litters Affected (#)	0	0
Total	0	0
Mean (+)	0.0	0.0
Standard Deviation	0.0	0.0
N	8	10
Living Pups and First Litter Check
% of Males/Females (#)	46/54	46/54
Total	84	124
Mean (+)	10.5	12.4
Standard Deviation	3.2	2.2
N	8	10
Postnatal Loss
% of Living Pups	1.2	0.0
Litters Affected (#)	1	0
Total (#)	1	0
Mean (+)	0.1	0.0
Standard Deviation	0.4	0.0
N	8	10
Culled Pups
Total	23	44
Living Pups Day 4 P.P.
Total	60	80
Mean (+)	7.5	8.0
Standard Deviation	1.4	0.0
N	8	10
Breeding Loss Days 5-13 P.P.
% of Living Pups at Day 4 P.P.	1.7	0.0
Litters Affected (#)	1	0
Total	1	0
Mean (+)	0.1	0.0
Standard Deviation	0.4	0.0
N	8	10
Living Pups Day 13 P.P.
% of Males/Females (#)	46/54	45/55
Total	59	80
Mean (+)	7.4	8.0
Standard Deviation	1.4	0.0
N	8	10
*100+/++ Steel-test significant at 5% (+) or 1% (++) level # / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

Developmental Data
	Group 1 (Control)	Group 4 (1000 mg/kg)
Total number of offspring born	84	124
Total number of uterine implantation sites	100	134
Number of live offspring on Day 1 (after littering)	84	124
Number of live offspring on Day 4 (before culling)	83	124
Number of live offspring on Day 4 (after culling)	60	80
Number of live offspring on Day 13 after littering	59	80
Post-implantation survival index (%)	84	93
Live birth index (%)	100	100
Viability index (%)	99	100
Lactation index (%)	98	100

Body Weights of Pups (Grams)
Day	Sex		Group 1 (Control)	Group 4 (1000 mg/kg)
1	M	Mean	6.5	6.6
		St. Dev.	0.8	0.7
		N	8	10
	F	Mean	6.3	6.3
		St. Dev.	0.8	0.6
		N	8	10
	M+F	Mean	6.4	6.4
		St. Dev.	0.7	0.6
		N	8	10
4	M	Mean	10.3	9.9
		St. Dev.	1.5	1.3
		N	8	10
	F	Mean	10.0	9.4
		St. Dev.	1.6	1.2
		N	8	10
	M+F	Mean	10.1	9.6
		St. Dev.	1.5	1.2
		N	8	10
7	M	Mean	17.2	16.4
		St. Dev.	1.7	1.6
		N	8	10
	F	Mean	17.0	15.6
		St. Dev.	2.1	1.4
		N	8	10
	M+F	Mean	17.0	16.0
		St. Dev.	1.8	1.4
		N	8	10
13	M	Mean	33.6	31.1
		St. Dev.	3.1	2.6
		N	8	10
	F	Mean	33.2	30.0*
		St. Dev.	3.5	1.8
		N	8	10
	M+F	Mean	33.3	30.5*
		St. Dev.	3.1	2.2
		N	8	10
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Historical Body Weights of Male Pups (Grams)
Day 1	Mean	6
	Standard Deviation	0.07
	N	2590
	P5	5.3
	P95	7.6
Day 4	Mean	10
	Standard Deviation	1.3
	N	2575
	P5	7.7
	P95	11.8
Day 7	Mean	16
	Standard Deviation	1.8
	N	1724
	P5	13.3
	P95	19.1
Day 13	Mean	31
	Standard Deviation	2.7
	N	1700
	P5	26.4
	P95	35.2

Historical Body Weights of Female Pups (Grams)
Day 1	Mean	6
	Standard Deviation	0.7
	N	2623
	P5	5.0
	P95	7.3
Day 4	Mean	9
	Standard Deviation	1.3
	N	2602
	P5	7.3
	P95	11.3
Day 7	Mean	16
	Standard Deviation	1.8
	N	1765
	P5	12.8
	P95	18.5
Day 13	Mean	30
	Standard Deviation	2.7
	N	1739
	P5	25.7
	P95	34.5

Anogenital Distance and Nipple Retention
F0- Generation - Lactation		Group 1 (Control)	Group 4 (1000 mg/kg)
Anogenital Dist. Male (mm)	Mean	2.66	2.56
	St. Dev.	0.16	0.17
	N	6	10
Anogenital Dist. Female (mm)	Mean	1.08	1.10
	St. Dev.	0.04	0.08
	N	6	10
Number of nipples	Mean	0.00	0.00
	Median (+)	0.00	0.00
	N	8	10
# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level
+/++ Steel-test significant at 5% (+) or 1% (++) level

Corrected Anogenital Distance
		Group 1 (Control)	Group 4 (1000 mg/kg)
PND 1
Norm anog dist – Male	Mean	1.42	1.36
mm	St. Dev.	0.06	0.06
	N	6	10
Norm anog dist - Female	Mean	0.59	0.60
mm	St. Dev.	0.02	0.05
	N	6	10

Clinical Biochemistry Summary – Male Pups
		Group 1 (Control)	Group 4 (1000 mg/kg)
Pups (PND 14-16)
Total T4	Mean	6.22	7.3
ug/dL	St. Dev.	0.91	0.99
	N	8	10

Clinical Biochemistry Summary – Female Pups
		Group 1 (Control)	Group 4 (1000 mg/kg)
Pups (PND 14-16)
Total T4	Mean	5.61	6.48
ug/dL	St. Dev.	0.82	0.68
	N	8	10

Number of animals with Microscopic findings by organ - Males
	Group 1 (Control) (10 animals)	Group 2 (100 mg/kg) (10 animals)	Group 3 (300 mg/kg) (10 animals)	Group 4 (1000 mg/kg) (10 animals)
Thyroid Gland	10	-	-	10
N.A.D.	7	-	-	6
Hypertrophy foll	3	-	-	4
Grade 1	3			4
Seminal Vesicles	10	-	-	10
N.A.D.	10	-	-	10
Coagulating Glands	10	-	1	10
N.A.D.	10	-	1	10
Prostate Gland	10	-	-	10
N.A.D.	9	-	-	9
Infiltrate inflamm.	1	-	-	1
Grade 1	1	-	-	1
Testes	10	-	-	10
N.A.D.	10	-	-	10
Epididymides	10	-	-	10
N.A.D.	10	-	-	7
Sperm Granuloma	-	-	-	3
Grade 2	-	-	-	2
Grade 3	-	-	-	1
Mandib. Lymph Nodes	-	-	1	-
Hypertrophy Lymphoid	-	-	1	-
Grade 1	-	-	1	-
Iliac Lymph Node	-	-	-	1
Hyperplasia lymphoid	-	-	-	1
Grade 2	-	-	-	1
Eyes	1	-	-	1
Hemorrhage, retro	1	-	1	-
Grade 2	-	-	1	-
Grade 3	1	-	-	-

Number of animals with Microscopic findings by organ - Females
	Group 1 (Control) (10 animals)	Group 2 (100 mg/kg) (10 animals)	Group 3 (300 mg/kg) (10 animals)	Group 4 (1000 mg/kg) (10 animals)
Thyroid Gland	10	-	2	10
N.A.D.	9	-	2	9
Hypertrophy foll	1	-	-	1
Grade 1	1	-	-	1
Ovaries	10	-	-	10
N.A.D.	10	-	-	10
Uterus	3	1	-	-
N.A.D.	1	-	-	-
Cyclic dilation	2	1	-	-
Cervix	3	-	-	-
N.A.D.	3	-	-	-
Vagina	3	-	-	-
N.A.D.	1	-	-	-
Cycle: Proestrus	1	-	-	-
Cycle: Estrus	1	-	-	-
Mandib. Lymph Nodes	4	-	-	-
Congestion	4	-	-	-
Grade 2	2	-	-	-
Grade 3	2	-	-	-
Adrenal Glands	-	-	1	1
N.A.D.	-	-	1	1
Thymus	-	-	1	-
Congestion	-	-	1	-
Grade 2	-	-	1	-

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

The reproductive/developmental and repeated dose toxicity of the test article was evaluated in male and female Wistar Han rats. The study was conducted according to OECD 421 in compliance with OECD GLP regulations. Rats (10/sex/dose) were dosed with 0 (control), 100, 300, or 1000 mg/kg/day via oral gavage for a minimum of 28 days. The following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight and food consumption, estrous cycle determination, measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development, mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)). Due to a dosing error occurring during the pre-mating period of the study, the animals of low and mid dose groups had received at least five 5 times less than intended. The results of these two groups were, therefore, excluded from interpretation. No parental toxicity was observed at the highest dose tested (1000 mg/kg/day). No adverse treatment-related changes were noted in any of the parameters investigated in this study (clinical appearance, body weight, food consumption, T4 thyroid hormone levels (in males only), macroscopic examination, organ weights, and microscopic examination. No treatment-related changes were noted in any of the reproductive parameters investigated (mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs). No treatment-related changes were noted in any of the developmental parameters investigated in this study (gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination). Based on the results of the study, the parental and reproductive/developmental No Observed Adverse Effect Level (NOAEL) is 1000 mg/kg/day.