cyclohexadecanone

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Feb - 25 May 2017

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: July 29, 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

438-930-8

EC Name:

-

Cas Number:

2550-52-9

Molecular formula:

C16H30O

IUPAC Name:

cyclohexadecanone

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

(Crl:CD(SD)), SPF

Details on species / strain selection:

Sprague-Dawley rats are commonly used in both the general systemic toxicity and reproductive and developmental toxicity studies with a large historical control data base. In addition, the rat is a required species in the regulatory guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10 weeks (males) and 12 (females)
- Weight at study initiation: Males: 336.3–388.7 g; Females: 230.1–284.9 g
- Housing: dosing period: 1 animal per cage; mating: 1:1; lactation: neonates were kept with the dam; animals were kept in stainless wire mesh cages (260W x 350D x 210H mm) and in polycarbonate cages (260W x 420L x 180H mm)
- Diet: Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C (Envigo RMS, Inc., U.S.A.), ad libitum
- Water: tap water, filtered and irradiated by ultraviolet light, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2–24.2
- Humidity (%): 38.7–60.1
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of the test substance was weighed using an electronic balance and placed in a bottle. A small amount of vehicle, corn oil, was added and both materials were mixed. The test substance was dissolved by stirring and heating at approximately 70°C and then, the vehicle was gradually added to yield the desired concentration. The dosing formulations were stored in a refrigerator (4.4–5.8°C). These dosing formulations were used within 7 days.

VEHICLE
- Justification for use and choice of vehicle: Through the preliminary solubility test to determine the solubility and dispersion characteristics of the test substance, corn oil was selected as vehicle because the test substance was well dissolved in it.
- Amount of vehicle: 5 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 11 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as Day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the dosing formulations were conducted using Gas Chromatography (GC-2010 series, Shimadzu Corp., Japan). Samples were taken three times from the middle of each dosing formulation prior to dosing and analyzed for verification of dose level concentration.
The results of dose concentration analyses were determined to be in the range of 101.40–108.50%. These results were within the acceptable limits (±15% of nominal values).

As a result of homogeneity and stability analyses, the 0.2 and 200 mg/mL dosing solutions were confirmed to be homogenous and stable for 4 hours at room temperature and for 7 days under refrigeration.

Duration of treatment / exposure:

Males were dosed once daily for a total of 42 days (for 2 weeks prior to mating, during 2 weeks of mating and 14 days post-mating). Females were dosed once daily for 2 weeks prior to mating until Postpartum Day 13. Also, females showing no evidence of parturition signs were dosed until Gestation Day 25.

Frequency of treatment:

once daily/ 7 days/week

Details on study schedule:

not applicable for an OECD 421 study

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: According to the information provided by the sponsor, the NOAEL (No Observed Adverse Effect Level) in the 28-day repeated oral toxicity study was 1,000 mg/kg bw/day. Therefore, the high dose level was selected at 1,000 mg/kg bw/day. Then, the mid and low dose levels were selected at 300 and 100 mg/kg bw/day, respectively. The control group animals were dosed with the vehicle only with the same dose volume as the test substance-treated groups.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily (general condition and clinical signs) and twice daily for moribundity and mortality
- Cage side observations included: general condition and clinical signs, mortality/viability, abortion and pre-mature birth

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of males were recorded just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing period, the day prior to necropsy and on the day of necropsy (fasted body weights). Body weights of females were recorded just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing period (except mating period), on Gestation Days 0, 7, 14 and 20, on Postpartum Days 0, 4 and 13, and on the day of necropsy (fasted body weights). Fasted body weights recorded on the day of necropsy were presented, but were not included in the statistical analysis.

FOOD CONSUMPTION: Yes
- Food consumption of males was recorded just prior to dosing on Day 0 (one day before the first dosing), once a week during the dosing period and the day prior to necropsy. Food consumption of females was recorded just prior to dosing on Day 0 (one day before first dosing), once a week throughout the dosing period, on Gestation Days 0, 6, 13 and 19, on Postpartum Days 0, 3 and 12 and the day prior to necropsy. Residual feed was recorded on the next day. Food consumption was not recorded during mating. Individual food consumption was calculated by subtracting the amount of residual feed from the amount presented.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

The estrus cycle examination for females was conducted from dosing initiation day to confirmed copulation day and on the day of necropsy. Smears of vaginal mucosa were prepared in the morning daily. Prepared smears of vaginal mucosa were stained with Diff Quick stain (Hemacolor® for microscopy, Merck, Germany). Stained vaginal mucosa smears were examined using light microscopy. The estrus cycle is divided into four stages; proestrus, estrus, metestrus and diestrus. One estrus cycle is defined as the period between the day of estrus and the day prior to next estrus. Estrous cycle was calculated from dosing initiation day to the day before mating initiation.

Sperm parameters (parental animals):

Parameters examined in male parental generation:
testis weight, epididymis weight, histopathological examination of testis and epididymis especially focused on spermatogenesis and interstitial testicular cell structure

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded. Litters of 8 pups or less were not culled. When litters contained less than four pups of one sex, pups of the opposite sex were added to make a total of 8 pups.

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, anogenital distance (AGD), presence of nipples in male pups

GROSS EXAMINATION OF DEAD PUPS: yes, for external abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on Day 43
- Maternal animals: All surviving animals on Postpartum Day 14; Non-delivered females were sacrificed on Gestation Day 26. Dams, whose pups were all dead, and animals found dead were sacrificed and examined as soon as possible.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the external surfaces and internal organs

ORGAN WEIGHTS
Paired organs (#) were weighed together. Animals were fasted overnight prior to necropsy and body weights were recorded on the day of necropsy. Organs were weighed and organ-to-body weight ratios of the following were calculated:
testis#, prostate and seminal vesicle with coagulating gland, thyroid#*, epididymis

* This organ was weight after fixation.

HISTOPATHOLOGY
At necropsy, the following organs and tissues with gross abnormalities were harvested and fixed in 10% neutral buffered formalin, except for the testes and epididymides which were fixed in Davidson fixative, and then stored in 10% neutral buffered formalin:
testis, prostate, ovary, vagina, organs and tissues with gross lesions, epididymis, seminal vesicle with coagulating gland, uterus and cervix, thyroid

Histopathological examination was carried out on the ovary, testis, epididymis, and other organs and tissues with gross abnormalities as follows.
- Control, low, mid and high dose groups
(especially focused on spermatogenesis and interstitial testicular cell structure)
- Animals found dead during the study
- Organs and tissues showing gross and macroscopic lesions at necropsy were examined.

THYROID HORMONE ANALYSIS
Blood samples were taken from all adult males and dams at termination.
Animals were fasted for more than 18 hours before necropsy and anesthetized with isoflurane. Blood samples were collected from the abdominal aorta in a vacutainer. T4 was analyzed in serum of adult males using an immunoassay analyzer.

Postmortem examinations (offspring):

SACRIFICE
- Pups were sacrificed after external examination on PND 4 and 13.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external examinations.

HISTOPATHOLOGY / ORGAN WEIGTHS
The thyroid from one male and one female pup per litter were preserved in 10% neutral buffered formalin on PND 13.

THYROID HORMONE ANALYSIS
Blood samples were taken from at least two pups per litter on PND 4 and 13, each (If the number of pups allows, from pups available above the culling target (8 pups/litter), from at least two pups per litter on both PND 4 and 13 otherwise. When only one pup was available above the culling target, only one pup was used for the determination of PND 4. When the litter size was below the culling target, blood collection was not carried out on PND 4.)
Animals were fasted for more than 18 hours before necropsy and anesthetized with isoflurane. Blood samples were collected from the heart (PND 4), abdominal vein or abdominal aorta (PND 13) in a vacutainer. Blood samples of pups on PND 4 were pooled by litter. Serum samples from the pups on PND 13 were analyzed for T4 using an immunoassay analyzer.

Statistics:

The statistical analysis of this study was conducted using the SAS program (SAS® 9.3, SAS Institute Inc., U.S.A.). For the data including body weights, food consumption, estrous cycle, mating period, gestation period, post-implantation loss, body weights of pups, birth and survival rates of pups, AGD index, nipple number, thyroid hormone value, organ weights, Bartlett’s test was conducted to analyze for homogeneity of variance (significance level: 0.05). One-way analysis of variance (ANOVA) test was employed on homogeneous data, then, if significant (significance level: 0.05), Dunnett’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Kruskal-Wallis test was employed on heterogeneous data, then, if significant (significance level: 0.05), Steel’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). The data of mating index, fertility index and other data associated with gestation were analyzed utilizing Fisher’s exact test (significance levels: 0.05 and 0.01).

Reproductive indices:

Mating index (%) = (number of females with confirmed mating / number of females placed with males) x 100
Mating period = Day of mating confirmed - Day of initial mating (based on dosing day)
Gestation period = Day 0 post partum - Day 0 of gestation (based on dosing day)
Male fertility index (%) = (number of males impregnating a female / number of males with confirmed mating) x 100
Female fertility index (%) = (number of pregnant females / number of females with confirmed mating) x 100
Gestation index (%) = (number of females with live pups / number of pregnant females) x 100
Post-implantation loss (%) = [(number of implantations - number of live pups) / number of implantations] x 100

Offspring viability indices:

Live birth index (%) = (number of live pups on postnatal Day 0 / number of implantations) x 100
Viability index on postnatal Day 0 (%) = (number of pups born alive on postnatal Day 0 / total number of pups born) x 100
Viability index on postnatal Day 4 (%) = (number of pups surviving on postnatal Day 4 / number of pups born alive on postnatal Day 0) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Surviving animals:
In the 300 and 1,000 mg/kg bw/day groups, salivation and soft stool were observed in males. Salivation or soft stool was observed in three males at 300 mg/kg bw/day from Day 22 to the end of dosing. Salivation or soft stool was frequently observed in all males at 1,000 mg/kg bw/day from Day 7 to the end of dosing.
Also, salivation was frequently observed in six females at 300 mg/kg bw/day from Day 7 of pre-mating period to parturition day, and salivation or soft stool was frequently observed in ten females at 1,000 mg/kg bw/day from Day 7 of pre-mating period to PPD 11. Vaginal bleeding, prolonged gestation (Gestation day,GD 25), delayed delivery, stillbirth and dystocia were observed in one female at 1,000 mg/kg bw/day on PPD 0. The numbers of dams whose pups were all dead (including stillbirth) were 1, 1, 4 and 2 in the 0 (control), 100, 300 and 1,000 mg/kg bw/day groups, respectively.

Dead animals:
Signs of soiled perineal region and a decrease in locomotor activity was observed in one female at 100 mg/kg bw/day before death occured (PPD 1). Two dams of 300 and 1000 mg/kg bw/day groups which had died on GD 24 and PPD 3, respectively, showed salivation from GD 5 to GD 23 and showed soft stool from GD 16 to GD 20.

Salivation and soft stool were considered to be test item related and were accompanied by an increase in food consumption during gestation but not causing effects on the body weights changes in the dams. Dystocia, stillbirth, prolonged gestation, delayed delivery and an increase of dams whose pups were all dead after parturition were considered to be treatment related. Other clinical signs observed in this study were considered to be incidental since they were transient and showed no dose-dependency.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

In the 100, 300 and 1,000 mg/kg bw/day groups, one female per group was found dead during the parturition or postpartum period.
One female was dead at 100 mg/kg bw/day on Postpartum Day (PPD) 1 with signs of soiled perineal region and a decrease in locomotor activity before death. No other test substance-related adverse effects were found in other parameters at 100 mg/kg bw/day. Also, one female was dead at 300 mg/kg bw/day during parturition due to dystocia on Gestation day 24 (prolonged gestation). One female was dead at 1,000 mg/kg bw/day without abnormal clinical signs on PPD 3.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no significant difference in the body weight in males at 100, 300 and 1,000 mg/kg bw/day.
A statistically significant (p<0.01) increase in the body weight was noted in females at 300 mg/kg bw/day on PPD 13. This change was not considered to be a test substance-related effect since it was transient and without dose-dependency.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A statistically significant (p<0.05, p<0.01) increase in food consumption was noted in males at 1,000 mg/kg bw/day (Day 36 and Day 42), and in females at 300 (GD 20 and PPD 13) and 1,000 mg/kg bw/day (GD 14 and GD 20). Eventhough, these differences did not result into body weight changes in both sexes, they are likely a compensatory behaviour due to the soft stool observed.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Dead animals:
The cause of death of the dams was not clear in histopathological examination. The gross findings observed in the one dam of the high dose group were in concordance with microscopically observed erosion in the stomach, atrophy of the thymus and white pulp (lymphoid) atrophy of the spleen. These findings were deemed to be incidental or stress-related, and to be not related to cause of death. Besides, the ovarian morphology of the three dams found dead was considered to be within normal variation.

Surviving animals:
There were no test substance-related changes in the testis, epididymis and ovary in males on Day 43, females on PPD 14, females whose pups were all dead and non-pregnant females. The macroscopic findings observed in one male corresponded to tubular atrophy in the testis and reduced sperm in the lumen of the epididymal duct, however it was considered to be spontaneous due to its isolated frequency. Atrophy of the ovary was seen in one female of the high dose group. This finding was also found in isolated frequency, and deemed to be age-related finding within the range of normal for SD rats since the animal was non-pregnant female.
In addition, obstructive nephropathy was observed in two dams whose pups were all dead. Observed tubular obstruction was deemed to be attributable to intratubular cell debris which might be produced by previous cellular damage. In one dam, nephropathy was accompanied by intratubular hemorrhage in the kidney, and it was correlated with black urine in the urinary bladder. These findings were observed with low incidence; did not show dose-response relationship; or could occur as idiopathic postpartum renal failure associated with preeclampsia/eclampsia, thus they were considered to be not associated with the test substance. All other microscopic findings seen in the organs and tissues with gross lesions were considered to be incidental or stress-related, and to be unrelated to the test substance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

THYROID HORMONE ANALYSIS:
There were no test substance-related changes in thyroid hormone (T4) levels of F0 males at 100, 300 and 1,000 mg/kg bw/day.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The estrous cycle lengths of females in the 0 (control), 100, 300 and 1,000 mg/kg bw/day groups were 4.1, 4.1, 4.0 and 4.4 days, respectively. There was no statistically significant difference in any dosing group. The estrous cycle of females on the day of necropsy was all diestrus except non-pregnant females which showed metestrus, proestrus or estrus.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Reduced sperm in the lumen of the epididymal duct of one male, however it was considered to be spontaneous due to its isolated frequency.
For further information see ´histopathological findings´.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating and gestation:
In the 0 (control), 100, 300 and 1,000 mg/kg bw/day dose groups, the mating periods were 2.9, 2.1, 1.8 and 2.0 days, the mating index was 100% in all dosing groups, the gestation periods were 22.4, 22.3, 22.5 and 22.4 days, and the fertility indices in both sexes were 91.7, 100.0, 100.0 and 75.0%, respectively. There was no statistically significant difference in the test substance dosing groups. Fertility index at 1,000 mg/kg bw/day was decreased due to three non-pregnant females. However, it was not considered to be a test substance-related effect because non-pregnancy of two females was related to the gross and histopathological lesions in the reproductive organs of one male and one female which were considered to be spontaneous. The gestation indices were 100.0, 100.0, 91.7 and 88.9%, respectively.

Delivery:
In the 0 (control), 100, 300 and 1,000 mg/kg bw/day dose groups, the post-implantation loss rates were 4.6, 16.7, 19.8 and 17.7%, the live birth indices were 95.4, 83.3, 80.2 and 82.3%, the mean litter sizes were 15.4, 12.3, 15.8 and 13.4. The numbers of abnormal delivery (dystocia or stillbirth) were 0, 0, 2 and 1 at 0, 100, 300 and 1,000 mg/kg bw/day, respectively. The sex ratios on PND 0 were 1.0, 1.1, 0.9 and 1.3, respectively. There was no statistically significant difference in the test substance dosing groups.
An increase of the post-implantation loss rate at 100 mg/kg bw/day and above and the decreases of live birth index, and abnormal deliveries at 300 and 1,000 mg/kg bw/day were considered to be related to poor health status of the respective dam and not directly associated with the test item treatment.
There is some indication that for most dams, whose pups were all dead, the dams were not in good health. Therefore, dystocia, stillbirth, prolonged gestation, delayed delivery and an increase of dams whose pups were all dead after parturition are not considered to be a direct effect of the test substance.

(see tables 1 and 2)

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects on reproduction up to and including the highest dose tested

2

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

3

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

For 0 (control), 100, 300 and 1,000 mg/kg bw/day dose groups the live birth indices were 95.4, 83.3, 80.2 and 82.3%, the viability indices on Postnatal Day (PND) 0 were 98.3, 91.7, 83.2 and 86.7%, and the viability indices on PND 4 were 89.2, 82.5, 77.1 and 83.3%, respectively. There was no statistically significant difference in the test substance dosing groups. However, the decreases of live birth index and viability index (PND 0 and 4) could be considered as indirect test substance-related effects.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test substance-related changes in the body weights of pups at 100, 300 and 1,000 mg/kg bw/day on PNDs 0, 4 and 13.

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:

no effects observed

Description (incidence and severity):

There were no test substance-related changes in Anogenital distance (AGD) index of pups at 100, 300 and 1,000 mg/kg bw/day. There was no nipple retention in male pups at 100, 300 and 1,000 mg/kg bw/day on PND 12.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no external findings in pups at 100, 300 and 1,000 mg/kg bw/day.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There were no test substance-related changes in thyroid hormone (T4) levels of pups of PND 13 at 100, 300 and 1,000 mg/kg bw/day.

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:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Target system / organ toxicity (F1)

Key result

Critical effects observed:

not specified

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

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

Dose response relationship:

no

Relevant for humans:

not specified

Any other information on results incl. tables

Table 1. Summary of Delivery and pups data

Dose/Group (mg/kg bw/day)		No. of implantations		Post-implantation loss rate (%)	Live birth index (%)	PND 0					PND 4		Viability index (%)		Sex ratio	Gestation index (%)	Delivery (no. of abnormality)
						No. of pups born			No. of live pups		No. of live pups
		left	right			live	dead	total	male	female	male	female	PND 0	PND 4
0 (Control)	Mean	7.4	8.5	4.6	95.4	15.1	0.3	15.4	7.7	7.4	7.4	6.3	98.3	89.2	1.0	100.0	0
	S.D.	2.0	1.4	5.0	5.0	1.6	0.6	1.6	3.5	3.0	4.0	3.6	3.9	29.9	85/81
	n	11	11	11	11	11	11	11	11	11	11	11	11	11	11
100	Mean	6.1	7.5	16.7	83.3	10.9	1.4	12.3	5.7	5.3	5.0	4.1	91.7	82.5	1.1	100.0	0
	S.D.	1.9	3.2	19.7	19.7	3.9	3.5	4.2	2.2	3.5	2.9	2.9	20.4	32.3	68/63
	n	12	12	12	12	12	12	12	12	12	11	11	12	11	12
300	Mean	7.9	8.3	19.8	80.2	13.0	2.8	15.8	6.3	6.7	5.4	6.1	83.2	77.1	0.9	91.7	2
	S.D.	2.0	2.1	29.9	29.9	5.2	5.5	1.9	3.4	3.0	3.5	3.7	31.4	41.0	69/74
	n	12	12	11	11	11	11	11	11	11	10	10	11	10	10
1000	Mean	8.3	6.0	17.7	82.3	12.6	0.9	13.4	7.0	5.6	6.0	5.6	86.7	83.3	1.3	88.9	1
	S.D.	2.3	2.8	31.7	31.7	5.4	1.7	4.1	3.7	3.0	3.0	3.2	32.8	35.0	63/50
	n	9	9	9	9	9	9	9	9	9	8	8	9	8	8

PND: Postnatal Day

- Post-implantation loss (%) = (No. of implantations – No. of live pups) / No. of implantations × 100

- Live birth index (%) = (No. of live pups on postnatal Day 0 / No. of implantations) × 100

- Viability index on postnatal Day 0 (%) = (No. of pups born alive on postnatal Day 0 / total No. of pups born) × 100.

- Viability index on postnatal Day 4 (%) = (No. of pups surviving on postnatal Day 4 / No. of pups born alive on postnatal Day 0) × 100

- Gestation index (%) = (Number of females with live pups / number of pregnant females) × 100

Table 2. Historical control data of delivery and pups (11 studies from 04/2016 -11/2017)

	No. of implantation		Post implantation loss rate (%)	Live birth index (%)	PND 0					PND 4		Viability index (%)		Sex ratio (male/female)	Gestation index (%)	Delivery (No. of abnormality)
	left	right			No. of pups born			No. of live pups		No. of live pups
					Live	dead	total	male	female	male	female	PND 0	PND 4
Mean	7.8	7.4	7.6	92.4	14.1	0.1	14.2	7.2	6.9	7.0	6.6	99.0	96.8	1.1	99.2	0.2
SD	0.6	0.8	2.7	2.7	1.0	0.1	1.0	0.7	0.6	0.7	0.5	0.8	2.9	0.1	2.7	0.4
Min	6.8	6.3	2.8	88.7	12.8	0.0	12.9	6.3	6.2	6.3	6.0	97.3	89.2	0.9	90.9	0.0
Max	8.5	8.5	11.3	97.2	16.1	0.4	16.1	8.4	7.7	8.4	7.6	100.0	100.0	1.3	100.0	1.0

PND: Postnatal Day

- Post-implantation loss (%) = (No. of implantations – No. of live pups) / No. of implantations × 100

- Live birth index (%) = (No. of live pups on postnatal Day 0 / No. of implantations) × 100

- Viability index on postnatal Day 0 (%) = (No. of pups born alive on postnatal Day 0 / total No. of pups born) × 100.

- Viability index on postnatal Day 4 (%) = (No. of pups surviving on postnatal Day 4 / No. of pups born alive on postnatal Day 0) × 100

- Gestation index (%) = (Number of females with live pups / number of pregnant females) × 100

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the NOAEL (fertility) was considered to be 1000 mg/kg bw/day for males since no effects occurred on fertility up to and including highest dose tested.
For females, NOAEL (fertiliy) was considered to be 100 mg/kg bw/day.
NOAEL for systemic toxicity was also determined to be 100 mg/kg bw/d based on clinical signs observed in the mid and high dose. One female died at 100 mg/kg bw/day on PPD 1 and increased post-implantation loss rate occured in dams at 100 mg/kg bw/day and above. No other test substance-related adverse effects were found in other parameters at 100 mg/kg bw/day. Prolonged gestation, dystocia, delayed delivery and stillbirth occurred at 300 mg/kg bw/day and above and are associated with the general health status of respective dams.
NOAEL for developmental toxicity of offspring was considered to be 100 mg/kg bw/day based on increased post-implantation loss rates and decreased live birth, viability indices (PND 0 and 4) and abnormal deliveries at 300 mg/kg bw/day and above.