Lithium phosphorodifluoridate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

Adopted on 29 July 2016

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

lithium(1+) (difluorophosphoryl)oxidanide

EC Number:

643-080-8

Cas Number:

24389-25-1

Molecular formula:

LiPO2F2

IUPAC Name:

lithium(1+) (difluorophosphoryl)oxidanide

Test material form:

solid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on species / strain selection:

This strain is widely used in reproduction/developmental toxicity studies using rodents, there is abundant historical data, and a large number of animals are available.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) ca. 10 wks
- Weight at study initiation: (P) Males: 362.2 - 418.3 g; Females: 217.9 - 258.4 g
- Housing: (1) For males and for females except the gestation and lactation periods; stainless hanging-type bracket cages (W × D × H: 226 × 346 × 198 mm), (2) For females during the gestation and lactation periods; polymethylpentene cages (W × D × H: 220 × 380 × 195 mm). Equipment for animal enrichment such as toys (alumina ball) and nesting materials and rest board was placed in the stainless cages. Nesting materials were placed in the polymethylpentene cages (including bedding). Toys and rest boards were exchanged once every two weeks or more frequently (6- to 14- day interval). Nesting materials were exchanged once a week or more frequently (1- to 7- day interval) when used in the stainless cages and concurrently with the cage exchange when used in the polymethylpentene cages.
- Diet (e.g. ad libitum) : ad libitum (rodent pellet diet)
- Water (e.g. ad libitum): ad libitum well water with sodium hypochlorite (free residual chlorine concentration: approx. 2 ppm)
- Acclimation period: 15 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 24.1°C
- Humidity (%): 43.2 - 71.0%
- Air changes (per hr): 10-20 per hour
- Photoperiod (hrs dark / hrs light): 12h light per day (7:00 - 19:00)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared under UV cut-off fluorescent light. The test substance was weighed in a glove box where air was replaced with nitrogen gas, and the relative humidity was confirmed to be below 30% with a hygrometer (actual value: 11% to 25%). The frequency of preparation of 1 and 3 mg/mL dosing formulations was once or twice every 6 or 7 days (permissible range: once or more every 8 days). The 0.3 mg/mL dosing formulation was prepared just before use. The vehicle (olive oil) was used as the dosing formulation for the control group.

(Control group dosing formulations)
The required amount of the vehicle was divided into polypropylene tubes for each dosing day.

(3 mg/mL dosing formulation)
(1) The test substance was weighed accurately and suspended by adding the vehicle gradually. Sonication (about 3 minutes) was repeated up to prepare a uniform suspension with an ultrasonic washing machine. Then the vehicle was added to make accurately the specified concentration (3 mg/mL) with a measuring cylinder.
(2) The dosing formulation after preparation was divided into polypropylene tubes for each dosing day.
(3) The tubes were filled with nitrogen gas.

0.3 and 1 mg/mL dosing formulations
(1) The 3 mg/mL dosing formulation was diluted with the vehicle to make 0.3 and 1 mg/mL dosing formulations. As for 0.3 mg/mL, the preparation was conducted before use.
(2) The dosing formulations after preparation were divided into polypropylene tubes for each dosing day.
(3) The tubes were filled with nitrogen gas.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of gestation (GD0)
- After successful mating each pregnant female was caged (how): individually (1/litter)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance formulations at 0.4, 4, and 100 mg/mL were confirmed to be stable and homogenous under refrigeration, in a dark place, in a tube filled with nitrogen gas for 8 days in another study.

At the first preparation, the sampling dosing suspensions (n=3) from 3 points (upper, middle, and lower layers) of the 1 and 3 mg/mL dosing formulations were confirmed for concentrations (actual value: 99.4 and 101.7%, within ±10% of
the nominal concentration) and their homogeneity (actual value: 1.9 and 0.7%, C.V. of the upper, middle, and lower layers, within ±10%). The measurement was conducted in accordance with the analysis protocol. For the 0.3 mg/mL dosing formulation, the concentration of the test substance was confirmed by recording the actual amount of the test substance weighed and the final volume of the dosing formulation at each preparation. Homogeneity of the test substance was confirmed by visual inspection.

Method of analysis: Ion Chromatography

Duration of treatment / exposure:

Males: From 14 days before mating (Days 1 to 15) until the day before necropsy through the mating period (35 days in total).
Females: From 14 days before mating (Days 1 to 15) until Day 12 of lactation (day of delivery was Day 0 of lactation) through the mating and gestation periods and delivery. Non-mating females and non-delivery females were maintained until the day before necropsy.

Frequency of treatment:

Daily

Details on study schedule:

The day of the start of dosing was designated as Day 1, and Days 1 to 7 as Week 1. Days 1 to 15 were before the mating period. For females, the day of successful copulation was designated as Day 0 of gestation (GD 0) and the day of delivery as Day 0 of lactation (LD 0).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control (Vehicle)

Doses / concentrations 2

Dose / conc.:

3 mg/kg bw/day (nominal)

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Middle dose

Doses / concentrations 4

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

12 per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose level selection:
The dose levels were set based upon the results of a dose Range-Finding Study (28 Days Repeated Oral Administration in Rats). In this study, 1 of 5 males at 40 mg/kg died on Day 29 (scheduled necropsy day). In addition, decreased body weight and food consumption were observed in males at 40 mg/kg, and some abnormalities were observed at necropsy in both sexes at 40 mg/kg. Therefore, the high dose level was set at 30 mg/kg which was less likely to die and was expected to develop some toxicity. The middle and low dose levels were set at 10 and 3 mg/kg/day, respectively, at a geometric ratio of about 3.

- Rationale for animal assignment (if not random):
On the day before the start of administration for both sexes, 9 females showing 5-day or more estrous cycles were excluded from the group assignment on the basis of the results of estrous cycle examination. The other animals were assigned to each group by the stratified randomization on the basis of the body weights (computer system; Provantis®, Instem LSS Limited). The animals weighing within ± 20% of the mean body weights (calculated for each sex) were used for this study.

Positive control:

Not applicable

Examinations

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed twice a day (before and after dosing) during the dosing period, and once a day in the other periods.

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed on Days 1, 8, 15, 22, 29, and 36. Females were weighed on Days 1, 8, 15, and/or 22 during the pre-mating period, GDs 0, 7, 14, and 20 during the gestation period, and LDs 0, 4, 7, and 13 during the lactation period. One female in which cohabiting was discontinued on Day 19 due to death of the partner male (hereinafter referred to as non-mating female) was also weighed on Days 22, 29, and 36.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OBSERVATIONS DURING DELIVERY AND LACTATION:
- All females with successful copulation were allowed natural delivery. The observation of delivery was conducted once daily (a.m. 9:00) from GDs 21 to 24. Females that
delivered their litter completely by 9:00 a.m. were judged as “delivered” on the corresponding day (the delivery day was regarded as LD 0), and when delivery was
completed at 9:00 or later, the following day was defined as LD 0.

HORMONE CONCENTRATION (TOTAL T4) ANALYSIS
- For parental animals, total T4 concentration was measured for males. Since changes attributable to the test substance treatment was observed in males at 30 mg/kg, females (LD13) were subjected to additional measurement.

Oestrous cyclicity (parental animals):

Vaginal smears were collected with swabs from all females in the morning (approximately same time every day) from the day of the start of dosing to the day of confirmed copulation. The obtained smears were collected on a plate for each animal, and stained with Giemsa. The estrous cycle was classified into diestrus (D), proestrus (P), estrus (E), and metestrus (M). The mean estrous cycle (number of days from the estrous period to the next estrous period) and the number of the estrous period during the test period were calculated.

Sperm parameters (parental animals):

Parameters examined male parental generations:
- testis weight, epididymis weight, sperm count in epididymides

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter) random removal of F1 offspring

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

HORMONE CONCENTRATION (TOTAL T4) ANALYSIS
The offspring were subjected to the measurement on PND 13 only. No measurements were conducted on PND 4 in any offspring.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on Day 36
- Maternal animals: All surviving animals on Lactation Day 13

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations thoracoabdominal organs/tissues were examined macroscopically.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring on post natal day (PND) 13.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations thoracoabdominal organs/tissues were examined macroscopically.

Statistics:

Statistical analysis (SA) was performed for the following data, except for the sex ratio, using a computer system (Provantis®, Instem LSS Limited). SAS 9.4 (SAS Institute Japan [CAC Croit Corporation]) was used for the sex ratio. In any case, two-tailed test was used, and levels of p<0.01 and p<0.05 were judged significant. SA of hormone concentration (total T4) was performed at the test site 2 using SAS 9.4 (EXSUS Version 8.1.0, SAS Institute Japan Ltd.). The data of offspring were analyzed on the basis of litter mean values. Moreover, the body weight and food consumption of non-pregnant female as well as the body weight of non-mating female on and after the start of mating were excluded from the evaluation.
Concerning dams with total litter loss, SA was conducted on the body weight and food consumption up to the day before total litter loss.

Multiple comparison test
The mean and SD were calculated and homogeneity of variance was tested by Bartlett’s method (p<0.05). When the groups were accepted as homogeneous, Dunnett’s multiple comparison test was used for comparison of the groups of data. When the groups of data were found to be heterogeneous by Bartlett’s test, Steel’s multiple comparison test was conducted.
Items: Body weights, food consumption, absolute and relative organ weights, estrous cycle, number of estrus, days until copulation, gestation length, number of implantations, number of delivered offspring, number of live offspring, body weight of offspring (both sexes) and AGD

Chi-square test
chi-square test was performed between the control and test item-treated groups.
Items: Copulation index, fertility index, gestation index, delivery index, and sex ratio

Wilcoxon’s rank sum test
Wilcoxon’s rank sum test was performed between the control and test item treated groups.
Items: Stillborn index, external anomaly index, external anomaly typing index, live birth index, viability index on Day 4, viability index on Day 13, and nipple development anomaly index

Reproductive indices:

Gestation index (%): (Number of pregnant animals delivered with live offspring / number of pregnant animals) × 100
Delivery index (%): (Total number of offspring at birth / number of implantations) × 100

Offspring viability indices:

Birth index (%): (Number of live offspring at birth / number of implantations) × 100
Stillborn index (%): (Number of stillborns / total number of delivered offspring) × 100
Viability index on Day 4 (%): (Number of live offspring on PND 4 / number of live offspring at birth) × 100
Viability index on Day 13 (%): (Number of live offspring on PND 13 / number of live offspring after culling) × 100
Sex ratio: (Number of male live offspring / number of female live offspring)
External anomaly index (%): (Number of offspring with external anomaly / number of observed offspring) × 100
External anomaly typing index (%): (Number of offspring with external anomaly by each type / number of observed offspring) × 100
Nipple development anomaly index (%): (Number of offspring with nipple development anomaly / number of observed offspring) × 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Two males at 30 mg/kg died on Days 21 and 19, respectively. For these animals, decrease in locomotor activity, perioral smudge, smudge of perinasal area, or bradypnea was observed from Day 14 until the day of death.
Three dams at 30 mg/kg died from GD 23 to LD 2. In one of these dams, salivation was observed on LD 0. In the other dams, no abnormal clinical signs were observed until the day of death.
In the surviving animals, salivation was sporadically observed after dosing in 6 males at 30 mg/kg on Day 12 or later and 3 females on Day 15 or later.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Two males at 30 mg/kg died on Days 21 and 19, respectively.
Three dams at 30 mg/kg died from GD 23 to LD 2.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males, body weight was significantly lower at 30 mg/kg than that in the control group on Days 15, 29, and 36.
In females, body weight was significantly lower at 30 mg/kg than that in the control group on LDs 4 and 7.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males, food consumption was significantly lower at 30 mg/kg than that in the control group on Day 2.
In females, food consumption was significantly lower at 30 mg/kg than that in the control group on LDs 1 and 4.

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:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

[Dead animals (2 males and 3 females at 30 mg/kg)]
In males, erosion in the mucosa of the glandular stomach was observed in 2 animals.
In females, erosion in the mucosa of the forestomach, erosion in the mucosa of the glandular stomach, and hyperplasia of squamous epithelium in the limiting ridge of the stomach were observed in 1, 2, and 3 animals, respectively.

[At the end of administration period]
In males, hyperplasia of squamous epithelium in the limiting ridge of the stomach was observed in 2 and 6 animals at 10 and 30 mg/kg, respectively.

Focal lymphocytic infiltration in the epididymis was observed in 3 and 1 animals of the control and 30 mg/kg groups, respectively; however, this change was not considered to be treatment-related because there was no dose-dependency. Atrophy of seminiferous tubule in the testis and decreased sperm in the lumen of the epididymis were observed in one male with small testis and epididymis observed at necropsy in the control group.

In females, hyperplasia of squamous epithelium in the limiting ridge of the stomach was observed in 1 animal at 30 mg/kg.

[Total litter loss (2 females at 30 mg/kg)]
Two females with litter loss showed erosion in the mucosa of the glandular stomach. One female also showed hyperplasia of squamous epithelium in the limiting ridge of the stomach.

[Non-mating and non-pregnant females]
No abnormalities were observed in any female.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

HORMONE CONCENTRATION (TOTAl T4) ANALYSIS
In males (F0), plasma total T4 concentration was significantly decreased at 30 mg/kg than that in the control group. The plasma total T4 concentration of the test item treated groups (Groups low-, middle-, and high-dose) was equivalent to the control group in parental animals [F0, female (LD 13)] and offspring [F1, PND 13].

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed in estrus count or estrous cycle in any test item-treated group.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

testis weight, epididymis weight - no statistical significant difference between teratment groups and controls.
sperm count in the lumen epididymides - no effects on sperm count in treatment groups.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

No statistically significant differences were observed in the days until copulation, copulation index, or fertility index in any test item treated group. One female at 3 mg/kg did not become pregnant.
Accordingly, the copulation indices were 100% for the control and all test item-treated groups, and the fertility indices were 100%, 91.7%, 100%, and 100% for the control, 3, 10, and 30 mg/kg groups, respectively.

The total litter loss was observed in 2 dams at 30 mg/kg on LD 0. Their litter was all stillborn. Due to total litter loss, although there was no statistical significance, gestation index (77.8%) and delivery index (86.669%) were lower at 30 mg/kg than those in the control group. These values were outside the range of historical data at the test facility.

Historical data (2013 to 2016, 6 studies, 10 to 12 dams/study)
Gestation index Mean: 100%
Delivery index Mean: 91.868% to 94.255%

No statistically significant difference was observed in the gestation length or number of implantation between the control group and any test item treated group.

Details on results (P0)

As an effect of test item on parent animals, death occurred at 30 mg/kg. In addition, decreases in body weights, food consumption and plasma total T4 concentration were observed at 30 mg/kg, and histopathological thickening of limiting ridge in the mucosa of the forestomach, which was suggestive of mucosal irritation of test item, was observed at 10 and 30 mg/kg.

Total litter loss was observed in 2 dams at 30 mg/kg on the day of delivery. Due to the total litter loss observed in the above animals, trends toward low values in gestation index and delivery index were observed at 30 mg/kg. In addition, trends toward low values in the number of live newborns and birth index, and trend toward a high value in the stillborn index were observed at 30 mg/kg.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

gross pathology

histopathology: non-neoplastic

2

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Total litter loss observed at 30 mg/kg in 2 dams

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

External examination of offspring (PND 13): No abnormalities were observed in any offspring. Offspring with external anomalies were observed in 1 offspring (0.649%) at 3 mg/kg. With regard to individual types of anomalies, acaudate was observed in 1 offspring (0.649%) at 3 mg/kg; however, this change was not considered to be treatment-related because there was no dose-dependency.
AGD (Anogenital distance, PND 4): No statistically significant difference was observed in the AGD between the control group and any test item -treated group.
Nipple development (PND 12): No statistically significant difference was observed in nipple development anomaly index between the control group and any test item-treated group.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The total litter loss was observed in 2 dams at 30 mg/kg on LD 0. Their litter was all stillborn.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In male offspring, body weight was significantly lower on PNDs 7 and 13 at 30 mg/kg than that in the control group. In female offspring, body weight was significantly lower on PNDs 4, 7 and 13 at 30 mg/kg than that in the control group.

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

HORMONE CONCENTRATION (TOTAL T4) ANALYSIS
The plasma total T4 concentration of the test item treated groups (Groups low-mid and high) for offspring [F1, PND 13] - no treatment related effects.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Although there was no statistical significance, the number of live newborns (9.4) and birth index (74.103%) were lower, and stillborn index (22.222%) was higher at 30 mg/kg than those in the control group. These values were outside the range of historical data at
the test facility.
Historical data (2013 to 2016, 6 studies, 10 to 12 dams/study)
Number of live newborns Mean: 12.8 to 14.6
Birth index Mean: 90.049% to 93.702%
Stillborn index Mean: 0% to 1.818%

No statistically significant difference was observed in the number of litter, sex ratio, or viability index on Day 4 or 13 between the control group and any test item-treated group.
Offspring with external anomalies were observed in 1 offspring (0.649%) at 3 mg/kg.
With regard to individual types of anomalies, acaudate was observed in 1 offspring (0.649%) at 3 mg/kg; however, this change was not considered to be treatment-related because there was no dose-dependency.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Table 2: Gross pathology findings (P0)

Sex	Male	Female	Female
Dose (mg/kg)	30	30	30
Number of Animals	2	3	2
Reason for necropsy	Dead	Dead	Total Litter loss
Necropsy findings
Present	2	3	2
Stomach
Dark reddish change; mucosa, glandular stomach	2	0	0
Dark reddish patch; mucosa, glandular stomach	0	1	2
Thickening; limiting ridge, mucosa, forestomach	0	3	1
Abnormal contents; dark red	2	0	0
Duodenum
Abnormal contents; dark red	2	-	-
Jejunum
Abnormal contents; dark red	2	-	-
Ileum
Abnormal contents; dark red	1	-	-
Cecum
Abnormal contents; dark red	1	-	-

Table 3: Body weights (F1, lactation period)

Day(s) Relative to Littering (PND)			Control	3 mg/kg	10 mg/kg	30 mg/kg
Sum of pup count (female)	0		80	71	87	44
	4		80	70	85	37
	7		49	44	50	23
	13		49	44	50	23
Mean female pup BW/Litter	0	Mean	6.703	6.627	6.777	6.370
		SD	0.745	0.631	1.035	1.151
		N	12	11	12	7
	4	Mean	11.182	11.264	10.853	9.241*
		SD	1.478	1.401	1.703	1.556
		N	12	11	12	6
	7	Mean	17.991	18.380	17.614	13.757**
		SD	1.834	1.972	1.841	3.010
		N	12	11	12	6
	13	Mean	34.099	34.448	33.103	27.736**
		SD	2.026	2.376	2.141	4.699
		N	12	11	12	6
Sum of pup count (male)	0		72	69	67	41
	4		72	69	67	39
	7		47	43	44	25
	13		47	43	44	25
Mean male pup BW/Litter	0	Mean	7.062	7.000	7.189	6.851
		SD	0.762	0.789	1.001	1.177
		N	12	11	12	7
	4	Mean	11.737	11.789	11.494	9.918
		SD	1.398	1.518	1.789	2.059
		N	12	11	12	6
	7	Mean	19.037	18.524	18.524	14.701*
		SD	1.729	1.967	1.967	3.199
		N	12	12	12	6
	13	Mean	35.694	34.549	34.549	29.208*
		SD	2.062	2.396	2.398	4.643
		N	12	12	12	6

* d-test: Dunnett 2-sided p<0.05

** d-test: Dunnett 2-sided p<0.01

Table 4: mean plasma total T4 hormone measurements

Group	Dose (mg/kg day)	Total T4 (nmol/L)
		P0, male	P0, female	F1, PND13
Control	0	40.4 ± 3.4	22.8 ± 4.8	47.6 ± 5.1
Low dose	3	39.4 ± 5.2	20.2 ± 2.9	50.6 ± 5.5
Middle dose	10	38.6 ± 5.8	24.1 ± 5.7	49.9 ± 6.7
High dose	30	34.5 ± 7.1*	23.7 ± 4.6	44.7 ± 12.4

* P<0.05: Significantly different from control group

Applicant's summary and conclusion

Conclusions:

The NOAELs for this study were 10 mg/kg/day (reproductive and developmental toxicity).