Polyamide wax

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2018-05-23 to 2018-08-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

yes

Remarks:

: see chapter 'any other information on materials and methods' section 7.8.2 /1

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

434-430-9

EC Name:

-

Molecular formula:

not applicable: UVCB substance

IUPAC Name:

12-hydroxy-N-[6-(12-hydroxyoctadecanamido)hexyl]octadecanamide

Test material form:

solid: particulate/powder

Details on test material:

Chemical registery number : 434-430-9
Chemical name : 12-hydroxystearic acid, reaction products with hexamethylene diamine

Specific details on test material used for the study:

Purity: Considered 100% (UVCB compound)
Correction factor: No correction factor
Expiry date: 24 April 2020

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Janvier, le Genest-Saint-Isle, France.
- Age: at the beginning of the treatment period, the females were 10-11 weeks old
- Mean body weight: at the beginning of the treatment period, the females had a mean body weight of 280 g (range: 230 g to 343 g).
- Fasting period before study: no
- Housing: the animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm2)
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for a period of at least 4 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 02 July 2018 to 29 August 2018.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION
The test item was administered as a suspension in the vehicle. The test item was ground to a fine powder, using a mortar and pestle, and then mixed with the required quantity of vehicle.
The test item dose formulations were prepared daily and stirred at least 30 minutes before administration. They were delivered to the study room at room temperature.
This preparation process was validated for a range of concentrations covering the lowest and highest concentrations used in this study.


VEHICLE
- Justification for use and choice of vehicle (if other than water): homogeneous suspensions were obtained with corn oil as a vehicle
- Concentration in vehicle: 20, 60 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: High Performance Liquid Chromatography with tandem Mass Spectrometry detection (LC/MS-MS)
Test item concentrations: the test item concentrations in the administered dose formulations analyzed in study Weeks 1, 2 and 3 of the treatment period (including the treatment period of additionnal females), were within an acceptable range of variation (-10.2% to +13.0%) when compared to the nominal values (± 15% required).
Homogeneity: The dose formulations containing the test item and prepared at 10 mg/mL and 200 mg/mL in corn oil were found to be homogeneous at room temperature and protected from light.
Stability: UVCB, dose formulation prepared daily

Details on mating procedure:

The females were mated at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was designated as Day 0 p.c.

Duration of treatment / exposure:

Days 6 to 20 post-coitum

Frequency of treatment:

Daily

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:

24 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for dose selection:
The dose levels were selected on the basis of the results of a 4-week toxicity study performed in CD rats by oral route. In this study, it was concluded that 1000 mg/kg/day represented the No Observed Effect Level (NOEL) for the test substance.
Therefore, 1000 mg/kg/day was selected as the high-dose level. The low-dose and mid-dose were selected using a ratio representing approximately a 3-fold interval (i.e. 300 and 100 mg/kg/day).

- Rationale for animal assignment: computerized stratification procedure.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: each animal was checked for mortality and morbidity once a day before and after the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

CLINICAL OBSERVATIONS:Yes
- Time schedule: from arrival, each animal was observed once a day as part of the routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

BODY WEIGHT:Yes
- Time schedule: the body weight of each female was recorded on Days 2, 4, 6, 9, 12, 15, 18 and 21 p.c.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food consumed by each female was recorded for the following intervals: Days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c.

WATER CONSUMPTION: No

POST-MORTEM MACOSCOPIC EXAMINATION:Yes
- Sacrifice on Day 21 post coitum
- Examined: principal thoracic and abdominal organs.

Ovaries and uterine content:

The ovaries and uterine content were examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
The weight of the gravid uterus was recorded for each pregnant female (with at least one live fetus) at hysterectomy.
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
- Number and distribution of dead and live fetuses,
- Number and distribution of uterine scars (uterine implantation without implant)
- Gross evaluation of placentas.

The following classification was used to record:
. uterine scar: uterine implantation without implant,
. early resorption: evidence of implant without recognizable embryo,
. late resorption: dead embryo or fetus with degenerative changes,
. dead fetus: dead fetus with no degenerative changes.

Fetal examinations:

- External examinations: Yes: all fetuses per litter
- Soft tissue examinations: Yes: half fetuses per litter
- Skeletal examinations: Yes: half fetuses per litter
- Head examinations: Yes: half fetuses per litter
- Other: number of dead and alive fetuses, fetal weight, fetal sex

Statistics:

Data were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous) or by Fisher’s exact probability test (proportions).

Indices:

The following parameters were calculated:
For each pregnant female:
- Body weight change for different intervals
- Net body weight (presented as carcass weight) = Body weight on Day 21 post-coitum - gravid uterine weight
- Net body weight change = Body weight on Day 21 post-coitum - body weight on Day 6 post-coitum - gravid uterine weight%

For each litter:
- Total number of resorptions = Sum of uterine scars + early resorptions + late resorptions
- Total number of dead fetuses = Sum of dead fetuses
- % of dead fetuses per litter = (Total number of dead fetuses / Number of implantation sites) x 100
- Total number of live fetuses = Sum of live male + live female fetuses
- % of live fetuses per litter = (Total number of live fetuses / Number of implantation sites) x 100
- % of pre-implantation loss = (Number of corpora lutea - Number of implantations / Number of corpora lutea) x 100
- % of post-implantation loss = (Number of implantation sites - Number of live fetuses / Number of implantation sites) x 100
- Average fetal body weight= Sum of individual fetal weights / Number of live fetuses

For each group:
- % of pre-implantation loss relative to the number of corpora lutea (mean of pre-implantation loss per litter)
- % of live fetuses and % of dead fetuses (relative to total number of fetuses)
- Mean % of male fetuses per litte
- Mean and standard deviations and % relative to the number of implantation sites: resorptions plus, uterine scars, uterine scars, early resorptions, late
resorptions,
- % of post-implantation loss relative to the number of implantation sites (mean of post-implantation loss per litter), % of dams affected.

Historical control data:

Cf attached document

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related clinical signs were observed.
Clinical signs (namely bent tail, cutaneous lesion and/or abnormal growth of teeth) were considered to be unrelated to the test item, as they were not dose-related and/or reported in isolated animals.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects on mean body weight and mean body weight change (see chapter 'any other information on results including tables' section 7.8.2 /2 for mean body weight and body weight changes tables).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no effects on mean food consumption (see chapter 'any other information on results including tables' section 7.8.2 /3 for mean food consumption tables).

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:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related macroscopic findings.
Enlarged placenta was noted in two fetuses from one female treated at 1000 mg/kg/day. These findings were considered to be related to the small size of the litter and of the fetuses which weighed 6.79 g and 6.84 g, respectively, and therefore were not considered to be test item-related.
The other changes (i.e. absence of uterine horn, placenta fused, with only one implantation site) were considered to be part of the spontaneous background in the rats of this strain as they were observed in control females.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There were no effects on mean gravid uterus and carcass weights and on mean net body weight change (see chapter 'any other information on results including tables' section 7.8.2 /4 for mean carcass weight, net body weight change and gravid uterus weight tables).

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no test item-related effects on pre and post implantation losses at any dose-level (see chapter 'any other information on results including tables' section 7.8.2 /5 for hysterectomy data).
Number of dams with pre-implantation loss: 17,11,14,11 at 0,100, 300 or 1000 mg/kg/day, respectively
Number of dams with post-implantation loss: 16, 12, 9, 9 at 0, 100, 300 or 1000 mg/kg/day, respectively

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

One female given 100 mg/kg/day had total resorption. As this finding occurred at the low-dose only, this was considered not to be test item-related (see chapter 'any other information on results including tables' section 7.8.2 /5 for hysterectomy data).

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no test item-related effects on early or late resorptions (see chapter 'any other information on results including tables' section 7.8.2 /5 for hysterectomy data).
Number of dams with resorptions: 16,12, 9, 9 at 0, 100, 300 or 1000 mg/kg/day, respectively.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no dead fetuses (see chapter 'any other information on results including tables' section 7.8.2 /5 for hysterectomy data).

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Number of pregnant females at hysterectomy: 24, 23, 22, 21 at 0, 100, 300 or 1000 mg/kg/day, respectively (see chapter 'any other information on results including tables' section 7.8.2 /5 for hysterectomy data).
As the test item was administered after implantation, the non-pregnant status for 1 female given 100 mg/kg/day, two females given 300 mg/kg/day and three females given 1000 mg/kg/day was considered to be fortuitous and not to be test item-related.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no effects on mean fetal body weight (see chapter 'any other information on results including tables' section 7.8.2 /6 for foetal body weight and sex ratio tables).

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no effects on sex ratio (% of males fetuses) (see chapter 'any other information on results including tables' section 7.8.2 /6 for foetal body weight and sex ratio tables).

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no changes in litter size and weights compared to controls

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-treatment related external malformations (see chapter 'any other information on results including tables' section 7.8.2 /7 for litter and fetal incidences of external malformations table).
In the 100 mg/kg/day group, one litter had one fetus with omphalocele. This external malformation was observed with an incidence similar to the Historical Control Data and without any dose relationship. Therefore, this was considered to be unrelated to the test item.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related skeletal malformations (see chapter 'any other information on results including tables' section 7.8.2 /8 for litter and fetal incidences of skeletal malformations table).
In the 300 mg/kg/day group, one litter had one fetus with cervical rib and one other litter had two fetuses with absent lumbar vertebra. As these skeletal malformations were observed without any dose relationship and with an incidence similar to the Historical Control Data, they were considered to be unrelated to the test item.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related soft tissue malformations (see chapter 'any other information on results including tables' section 7.8.2 /9 for litter and fetal incidences of soft tissue malformations table).
In the control group, one litter had one fetus with marked renal dilated pelvis and marked dilated ureter. These tissue malformations were observed with an incidence similar to the Historical Control Data.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

External variations:
There were no external variations.

Skeletal variations:
There were no test item-related skeletal variations (see chapter 'any other information on results including tables' section 7.8.2 /10 for litter and fetal incidences of skeletal variations table).
When compared with controls, variations were observed at a slightly higher fetal and/or litter incidence in the 300 and/or 1000 mg/kg/day group [i.e. incomplete ossification of supraoccipital, incomplete ossification of parietal, dumbbell ossification of thoracic vertebra(e) centrum, ossification point on lumbar vertebra(e), extra sternebral ossification site and incomplete ossification of rib and unossified forepaw proximal phalanx]. These variations did not impact mean fetal body weights and were observed with incidences similar or very close to the Historical Control Data and/or were not statistically significant.
As the other skeletal variations were not observed at the high-dose level or were noted at a similar or lower incidence than that recorded in controls, they were also considered to be unrelated to the test item treatment.

Visceral variations:
There were no test item-related soft tissue variations (see chapter 'any other information on results including tables' section 7.8.2 /11 for litter and fetal incidences of soft tissue variations table).
When compared with controls, dilated renal pelvis and ureter were observed at higher litter and fetal incidences from 300 mg/kg/day. As these variations were observed with incidences similar to the Historical Control Data and were not statistically significant, they were therefore considered to be unrelated to the test item.
As the other tissue variations were noted at a lower incidence than that recorded in controls, they were also considered to be unrelated to the test item.

Cartilage
There were no test item-related cartilage findings (see chapter 'any other information on results including tables' section 7.8.2 /12 for litter and fetal incidences of cartilage findings table).
In the 1000 mg/kg/day group and when compared with controls, there were decreases [i.e. cartilage of rib(s) present] or increases [i.e. cartilage of rib(s) absent, misshapen cartilage rib(s) and/or cartilage of forepaw proximal phalanx present] in the litter and fetal incidences of cartilage findings. These findings did not impact mean fetal body weights. They were not statistically significant and with an incidence similar or lower than the controls and/or Historical Control Data. They were therefore considered to be unrelated to the test item treatment.

Effect levels (fetuses)

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Table 7.8.2/ 2 : Mean body weight and body weight changes (g)

Dose level (mg/kg/day)	0	100	300	1000
Body weight (g)
Day 6 p.c.	281	281	281	280
	-	(0)	(0)	(0)
Day 9 p.c.	292	292	292	290
	-	(0)	(0)	(-1)
Day 12 p.c.	309	312	312	309
	-	(+1)	(+1)	(0)
Day 15 p.c.	328	333	332	329
	-	(+2)	(+1)	(0)
Day 18 p.c.	375	383	381	374
	-	(+2)	(+2)	(0)
Day 21 p.c.	425	434	434	422
	-	(+2)	(+2)	(-1)
Body weight change (g)
Days 6 - 9 p.c.	+11	+11	+11	+10
Days 9 - 12 p.c.	+17	+20	+20	+19
Days 12 - 15 p.c.	+19	+20	+20	+20
Days 15 - 18 p.c.	+46	+50	+49	+45
Days 18 - 21 p.c.	+51	+51	+52	+48
Days 6 - 21 p.c.	+144	+153	+153	+143
	-	(+6)	(+6)	(-1)

p.c.: post-coitum.

-: not applicable.

( ): in brackets, percentage difference vs.controls.

 

Table 7.8.2/3: Mean food consumption (g/animal/day)

Dose level (mg/kg/day)	0	100	300	1000
. Days 6 - 9 p.c.	21	21	23	21
. Days 9 - 12 p.c.	24	23	26	24
. Days 12 - 15 p.c.	26	26	27	26
. Days 15 - 18 p.c.	34	31	34	31
. Days 18 - 21 p.c.	29	29	31	29

p.c.: post-coitum.

 

Table7.8.2/4: Mean carcass weight, net body weight change and gravid uterus weight (g)

Dose level (mg/kg/day)	0	100	300	1000
Gravid uterus weight	98	106 (+8)	100 (+2)	97 (-1)
Carcass weight	328	328 (0)	333 (+2)	326 (-1)
Net body weight change from Day 6 p.c.	+47	+47	+52	+46

( ): in brackets, percentage difference vs. controls.

p.c.: post-coitum.

^(a): weights are rounded values.

Table7.8.2/5a: Pregnancy status

Dose level (mg/kg/day)	0	100	300	1000
Number of females	24	24	24	24
Non-pregnant females	0	1	2	3
Females with total resorption	0	1	0	0
Females with live fetuses at term	24	22	22	21

 

Table7.8.2/5b: Hysterectomy data

Dose level (mg/kg/day)	0	100	300	1000
Number of pregnant females at hysterectomy	24	23	22	21
Number of females with live fetuses at termination	24	22	22	21
Number of females with total resorption	0	1	0	0
Mean number of corpora lutea	14.0	15.2	13.7	13.1
Mean number of implantation sites	12.8	13.3	12.8	12.2
Mean pre-implantation loss (%)	9.2	11.2	6.5	7.3
Number of females with pre-implantation loss	17	11	14	11
Mean number of live fetuses	11.9	12.6	12.1	11.8
Dead fetuses (%)	0.0	0.0	0.0	0.0
Mean number of implantation scars	0.0	0.0	0.0	0.0
Mean number of early resorptions	0.7	0.7	0.7	0.4
Mean number of late resorptions	0.2	0.1	0.0	0.1
Mean number of resorptions + scars	0.9	0.7	0.7	0.5
Number of females with resorptions	16	12	9	9
Mean post-implantation loss (%)	7.4	9.4	5.4	3.7
Number of females with post-implantation loss	16	12	9	9

 

Table7.8.2/6: fetal body weight and sex ratio

Dose level (mg/kg/day)	0	100	300	1000
Mean fetal body weight (g)	5.88 -	5.82 (-1)	5.94 (+1)	5.92(+1)
Mean fetal body weight of males (g)	6.05 -	5.97 (-1)	6.09 (+1)	6.09 (+1)
Mean fetal body weight of females (g)	5.74 -	5.64 (-2)	5.75 (0)	5.78 (+1)
Mean percentage of male fetuses (%)	46.6	52.3	52.1	47.5

( ): in brackets, percentage difference vs.controls.

-: not applicable.

 

Table7.8.2/7: litter (L) and fetal (F) incidence (%) of external malformations

Fetal external malformations

Dose level (mg/kg/day)	0	100	300	1000	HCD
Dams with live fetuses, n	24	22	22	21	235
Live fetuses, n	286	290	266	247	2896
. omphalocele, L(F)	0 (0)	4.5 (0.3)	0 (0)	0 (0)	4.2 (0.3)^(a)
Litters affected, n (%)^(c)	0 (0)	1 (4.5)	0 (0)	0 (0)	8 (3.4)^(b)
Fetuses affected, n (%)^(c)	0 (0)	1 (0.3)	0 (0)	0 (0)	10 (0.3)^(b)

n: number.

HCD: Historical Control Data (control data collected from 11 studies covering a period ranging from August 2016 to December 2017.

^(a): upper litter (fetal) incidence per study; ^(b): mean number (percentage) of litters or fetuses affected.
^(c): all malformations combined.

 

Table7.8.2/11: litter (L) and fetal (F) incidence (%) of soft tissue variations

Dose level (mg/kg/day)	0	100	300	1000	HCD
Dams with live fetuses, n	24	22	22	21	235
Live fetuses, n	136	141	127	120	1385
. kidney: dilated pelvis, L(F)	16.7 (3.7)	13.6 (2.1)	22.7 (6.3)	28.6 (5.8)	30.8 (9.1)^(a)
. ureter: dilated, L(F)	29.2 (8.8)	18.2 (5.0)	40.9 (12.6)	33.3 (6.7)	48.7 (15.1)^(a)
Litters affected, n (%)^(c)	12 (50.0)	6 (27.3)	10 (45.5)	8 (38.1)	70 (29.8)^(b)
Fetuses affected, n (%)^(c)	18 (13.2)	10 (7.1)	21 (16.5)	11 (9.2)	132 (9.5)^(b)

n: number.

HCD: Historical Control Data (control data collected from 11 studies covering a period ranging from August 2016 to December 2017).

(a): upper litter (fetal) incidence per study; (b): mean number (percentage) of litters or fetuses affected.
(c): all variations combined.

 

Table7.8.2/9: litter (L) and fetal (F) incidence (%) of soft tissue malformations

Dose level (mg/kg/day)	0	100	300	1000	HCD
Dams with live fetuses, n	24	22	22	21	235
Live fetuses, n	136	141	127	120	1385
. kidney: marked dilated  pelvis, L(F)	4.2 (0.7)	0 (0)	0 (0)	0 (0)	4.2 (0.7)^(a)
. ureter: marked dilated,  L(F)	4.2 (0.7)	0 (0)	0 (0)	0 (0)	10.3 (2.2)^(a)
Litters affected, n (%)^(c)	1 (4.2)	0 (0)	0 (0)	0 (0)	7 (3.0)^(b)
Fetuses affected, n (%)^(c)	1 (0.7)	0 (0)	0 (0)	0 (0)	10 (0.7)^(b)

n: number.

HCD: Historical Control Data (control data collected from 11 studies covering a period ranging from August 2016 to December 2017).

^(a): upper litter (fetal) incidence per study; ^(b): mean number (percentage) of litters or fetuses affected.

^(c): all malformations combined.

 

Table7.8.2/12: litter (L) and fetal (F) incidence (%) of cartilage findings

Dose level (mg/kg/day)	0	100	300	1000	HCD
Dams with live fetuses, n	24	22	22	21	235
Live fetuses, n	149	149	139	127	1511
. cartilage of rib(s) present, L(F)	16.7 (4.7)	13.6 (2.0)	18.2 (2.9)	9.5 (1.6)	16.7 (3.8)^(a)
. cartilage of rib(s) absent,  L(F)	0 (0)	0 (0)	0 (0)	4.8 (0.8)	4.3 (0.6)^(a)
. cartilage of rib(s) misshapen, L(F)	4.2 (1.3)	4.5 (0.7)	4.5 (1.4)	4.8 (0.8)	0 (0)
. forepaw: cartilage of  proximal phalanx  present, L(F)	33.3 (13.4)	54.5 (14.1)	40.9 (15.8)	42.9 (13.4)	87.5 (50.0)^(a)
Litters affected, n (%)^(c)	21 (87.5)	20 (90.9)	19 (86.4)	19 (90.5)	128 (54.5)^(b)
Fetuses affected, n (%)^(c)	98 (65.8)	99 (66.4)	90 (64.7)	73 (57.5)	584 (38.6)^(b)

n: number.

HCD: Historical Control Data (control data collected from 11 studies covering a period ranging from August 2016 to December 2017).

^(a): upper litter (fetal) incidence by study; ^(b): mean number (percentage) of litters or fetuses affected.
^(c): all cartilage findings combined.

 

Table7.8.2/10: litter (L) and fetal (F) incidence (%) of skeletal variations

Dose level (mg/kg/day)	0	100	300	1000	HCD
Dams with live fetuses, n	24	22	22	21	235
Live fetuses, n	149	149	139	127	1511
. supraoccipital: incomplete  ossification, L(F)	0 (0)	4.5 (0.7)	0 (0)	9.5 (1.6)	8.3 (3.2)^(a)
. interparietal: incomplete  ossification, L(F)	0 (0)	9.1 (1.3)	9.1 (1.4)	9.5 (2.4)	21.1 (4.5)^(a)
. thoracic vertebra(e): dumbbell  ossification of centrum, L(F)	0 (0)	4.5 (0.7)	0 (0)	4.8 (0.8)	25.0 (4.7)^(a)[DP1]
. lumbar vertebra(e): ossification  point, L(F)	4.2 (0.7)	9.1 (1.3)	0 (0)	9.5 (1.6)	4.2 (0.6)^(a)
. extra sternebral ossification  site, L(F)	4.2 (0.7)	13.6 (4.0)	4.5 (0.7)	23.8 (3.9)	25 (6.0)^(a)
. rib: incomplete ossification, L(F)	4.2 (0.7)	4.5 (0.7)	9.1 (2.2)	9.5 (1.6)	15.8 (4.8)^(a)
. forepaw: unossified proximal  phalanx, L(F)	33.3 (13.4)	54.5 (14.1)	40.9 (15.8)	42.9 (13.4)	87.5 (50.0)^(a)
Litters affected, n (%)^(c)	21 (87.5)	20 (90.9)	20 (90.9)	19 (90.5)	194 (82.6)^(b)
Fetuses affected, n (%)^(c)	99 (66.4)	103 (69.1)	91 (65.5)	76 (59.8)	742 (49.1)^(b)

n: number.

HCD: Historical Control Data (control data collected from 11 studies covering a period ranging from August 2016 to December 2017).

^(a): upper litter (fetal) incidence by study; ^(b): mean number (percentage) of litters or fetuses affected.

^(c): all variations combined.

 

Table7.8.2/8 : litter (L) and fetal (F) incidence (%) of skeletal malformations

Dose level (mg/kg/day)	0	100	300	1000	HCD
Dams with live fetuses, n	24	22	22	21	235
Live fetuses, n	149	149	139	127	1511
. cervical vertebra(e): cervical rib, L(F)	0 (0)	0 (0)	4.5 (0.7)	0 (0)	0 (0)
. lumbar vertebra(e): absent, L(F)	0 (0)	0 (0)	4.5 (1.4)	0 (0)	5.3 (2.4)^(a)
Litters affected, n (%)^(c)	0 (0)	0 (0)	2 (9.1)	0 (0)	13 (5.5)^(b)
Fetuses affected, n (%)^(c)	0 (0)	0 (0)	3 (2.2)	0 (0)	15 (1.0)^(b)

n: number.

HCD: Historical Control Data (control data collected from 11 studies covering a period ranging from August 2016 to December 2017).

^(a): upper litter (fetal) incidence per study; ^(b): mean number (percentage) of litters or fetuses affected.

^(c): all malformations combined.

Applicant's summary and conclusion

Conclusions:

The No Observed Effect Level (NOEL) for maternal parameters and for embryo-fetal development was considered to be 1000 mg/kg/day in absence of test item treatment effects in the study.

Executive summary:

In a prenatal development toxicity study performed according to OECD 414 and in compliance with Good Laboratory Practice, the objective was to evaluate the potential toxic effects of the test substance on the pregnant female rat and on embryonic and fetal development, following oral administration (gavage) from Day 6 to Day 20 post-coitum, inclusive. 

Three groups of 24 time-mated female rats received the test substance at doses of 100, 300 or 1000 mg/kg bw /day from Day 6 to 20 post-coitum. A similarly constituted Control group received the vehicle, corn oil, at the same dose volume throughout the same period. Test substance concentration was checked four times in formulations given to the animals

Animals were killed on Day 21 of gestation for reproductive assessment and fetal examination.

Clinical observations, bodyweight and food consumption were monitored. Adult females were examined macroscopically at necropsy on Day 21 of gestationand the numbers of corpora lutea, implantations, early and late resorptions, and live and dead fetuses were recorded. All fetuses were weighed, sexed and examined for external, soft tissue and skeletal abnormalities.

The test item concentrations in the analyzed dose formulations were within an acceptable range of variation (between -10.2% and +13.0%) when compared to the nominal values (± 15% required).

At hysterectomy on Day 21 post-coitum., there were 24/24, 22/24, 22/24 and 21/24 pregnant dams with live fetuses in the groups treated at 0,100, 300 or 1000 mg/kg/day,respectively. The non-pregnant status of 1 female given 100 mg/kg/day, two females given 300 mg/kg/day and three females given 1000 mg/kg/day was not test-item related as the test item was administered after implantation.

There were no test item treatment-related effects in the dams in terms of mortality, clinical signs, necropsy findings, body weight, food consumption, carcass weight, gravid uterus weight, net body weight change, or hysterectomy data. There were no test item treatment-related effects in the litters in terms of sex ratio, fetal body weight, external, visceral and skeletal variations or malformations or cartilage findings.

The No Observed Effect Level (NOEL) for maternal parameters and for embryo-fetal development was considered to be 1000 mg/kg/day in absence of test item treatment effects in the study.