Reaction mass of Amines, coco alkyl and β-Alanine, N-(2-carboxyethyl)-, N-coco alkyl derivs. and β-Alanine, N-coco alkyl derivs.

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 July 2016 - 15 September 2016

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 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

22 January 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Amines, coco alkyl and ß-Alanine, N-(2-carboxyethyl)-, N-coco alkyl derivs. and ß-Alanine, N-coco alkyl derivs.

EC Number:

915-790-0

Molecular formula:

n/a

IUPAC Name:

Reaction mass of Amines, coco alkyl and ß-Alanine, N-(2-carboxyethyl)-, N-coco alkyl derivs. and ß-Alanine, N-coco alkyl derivs.

Test material form:

other: solid paste

Details on test material:

- Name: Reaction mass of Amines, coco alkyl and ß-Alanine, N-(2-carboxyethyl)-, N-coco alkyl derivs. and ß-Alanine, N-coco alkyl derivs.
- Synonyms: Amphoram CP1 without solvent ; Amphoram CP 1 ; Amphoram CP1
- Batch No.: EL1699 (also seen as EL 1699)
- Description: yellow to brown paste
- Storage condition: at room temperature
- Expiry date: 13 January 2019.

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 animals were 10-11 weeks old
- Mean body weight at the beginning of the treatment period: 286 g (range: 229 g to 337 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 4 or 5 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: 16 August 2016 to 15 September 2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING FORMULATIONS:
- Suspension in the vehicle
- Concentration in vehicle: 12, 34 and 100 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.

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: remained within an acceptable range of variation (-1.3% to +7.0%) when compared to the nominal values (± 15% required).
Homogeneity: The dose formulations containing the test item in corn oil at 0.2 mg/mL and 200 mg/mL were found to be homogeneous after preparation.
Stability: no stability available, dose formulations 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 p.c.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

500 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-level selection:
The dose-levels were selected in agreement with the Sponsor, on the basis of the results of the following previous study: preliminary non-GLP study of prenatal developmental toxicity by oral route (gavage) in rats where groups of 8 time-mated female Sprague-Dawley rats received the test item daily from Days 6 to 20 p.c. by gavage at 120, 300 and 600 mg/kg/day in corn oil. At 600 mg/kg/day, ptyalism, piloerection and round back were recorded for all or most females. Mean food consumption was reduced on Days 6 to 15 p.c. (from -30 to -38% vs. controls) and concomitantly slight mean body weight loss and then lower mean body weight gain were noted. On Days 18-21 p.c., low mean food consumption (-29% vs. controls) and low mean body weight gain were considered to be related to the lower mean fetal body weight (-21% vs. controls) seen at hysterectomy. The net body weight change from Day 6 p.c. was negative for half of the females while positive for all other females of the study.
At 300 mg/kg/day, ptyalism was noted in all females. Reduced mean food consumption (-19% vs. controls) was concomitant to slight mean body weight loss on Days 6 to 9 p.c.
At 120 mg/kg/day, there were no toxicologically significant effects.

Therefore, 500 mg/kg/day was selected as the high dose-level. The low-dose and mid dose were selected using a ratio representing an approximately 3-fold interval (i.e. 60 and 170 mg/kg/day).

- Rationale for animal assignment: computerized stratification procedure.

Examinations

Maternal examinations:

MORTALITY/MORBIDITY:
- Time schedule: each animal was checked once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

CLINICAL SIGNS:
- 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:
- 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:
- Time schedule: 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.

POST-MORTEM MACROSCOPIC EXAMINATION:
- 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, including:
- gravid uterus weight,
- number of corpora lutea,
- number and distribution of dead and live fetuses,
- number and distribution of early and late resorptions,
- number and distribution of uterine scars,
- number and distribution of implantation sites.
- gross evaluation of placentas

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: body weight, 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 exact probability test (proportions).

Indices:

% Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
% Post-implantation loss = 100 * (Number of implantation sites - Number of live fetuses) / Number of implantation sites

Historical control data:

see attached document.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See table 1.
All test item-treated females (except one at 60 mg/kg/day) had ptyalism for up to all or nearly all the treatment period. This test item-related finding was considered as non-adverse.

At 500 mg/kg/day:
- there were one female with several clinical signs (soft feces, soiled urogenital area, round back, piloerection, emaciated appearance) from Day 19, 20 or 21 p.c. From Day 15 p.c. until sacrifice, this female had no food intake and lost 15% of body weight,
- one female was emaciated on Day 21 p.c.; on Days 18 to 21 p.c. it had no food intake and lost 2% of body weight.
All these findings were similar to that recorded in the found dead female. Therefore, they were considered to be test item-related and adverse.

- one female had piloerection from Day 15 p.c. which could be related to the test item treatment in view of the clinical condition of the above-mentioned females,
- there were two females with loud or abdominal breathing on one day only which were considered incidental,
- dacryhorrhea and chromorhinorrhea recorded in isolated females were also considered to be incidental.

At 170 mg/kg/day, one female had loud breathing from Day 15 p.c. to the end of the study. Taking into account that respiratory difficulty was also observed in the found dead animal at the high-dose, a test item effect was not ruled out. However, its body weight gain and food consumption were not impacted.

Mortality:

mortality observed, treatment-related

Description (incidence):

One female given 500 mg/kg/day was found dead on Day 19 p.c.
From Day 15 p.c., emaciated appearance, round back, abdominal and/or loud breathing (observed before treatment on Day 15 p.c.), and/or dyspnea were observed. Ptyalism was noted from Day 10 p.c.
Its food consumption was reduced from Day 6 p.c. to become null before death. The female lost 16% of body weight from Days 12 to 18 p.c.
At necropsy, thymus and spleen were reduced in size likely related to its poor health condition of the female on the last days before death. There were 16 dead fetuses and 1 late resorption in the uterus.
In absence of microscopic examination, it was not possible to determine the cause of death. However, in view of similar general conditions in a few other females at the same dose, this death was considered to be test item-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See table 2.
At 500 mg/kg/day, mean body weight loss was recorded from Days 6 to 9 p.c. followed by mean body weight gains lower than controls. Mean body weight was decreased (down to -11% vs. controls, p < 0.001). These effects were considered to be test item-related. They correlated with the effects on mean carcass weight, mean net body weight change and mean fetal weight.

At 60 and 170 mg/kg/day, there were no effects on mean body weight and mean body weight change.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See table 3.
At 500 mg/kg/day and when compared with controls, there was a lower mean food consumption throughout the treatment period and mainly at the beginning of the study; this finding was considered to be test item related.

At 60 and 170 mg/kg/day, there were no effects on mean food consumption.

Food efficiency:

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):

See table 4.
None of the necropsy findings were ascribed to the test item treatment: they were noted in isolated cases, were not dose-related and/or were congenital anomalies.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

See table 5.
Mean gravid uterus and carcass weights and mean net body weight changes
At 500 mg/kg/day, all three parameters were lower than in controls and correlated with the effects seen on mean body weight seen previously and mean fetal body weight discussed in the next pages. These effects were considered to be test item-related and non-adverse (differences from controls not too severe).

At 60 and 170 mg/kg/day, there were no effects on mean carcass and gravid uterus weights and on mean net body weight change from Day 6 p.c.

Maternal developmental toxicity

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

See table 6.
There were no effects on mean hysterectomy data.
At 500 mg/kg/day, the minimally higher mean number of mean post-implantation loss when compared with controls was due to one female only and considered as incidental.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

All surviving pregnant females had live fetuses on Day 21 p.c.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

See table 6.
At 500 mg/kg/day, the minimally higher mean number of early resorptions when compared with controls was due to one female only and considered as incidental.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

See table 6.

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

All females were pregnant, except one at 60 mg/kg/day.
All surviving pregnant females had live fetuses on Day 21 p.c.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

Maternal abnormalities

Abnormalities:

not examined

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

See table 7.
At 500 mg/kg/day and when compared with controls, the lower mean fetal body weight was considered to be test item-related and adverse in view of the amplitude of difference from controls. The two lowest mean fetal body weight were for 2 dams with poor general condition by the end of gestation.

At 60 and 170 mg/kg/day, there were no effects on mean fetal body weight.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no effects on sex ratio.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

see table 8.
None of the malformations were considered to be test item-related: they were noted in isolated incidence, comparably to historical control data and were not dose-related.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

At 500 mg/kg/day, there was one fetus with a cervical rib.
At 170 mg/kg/day, there was one fetus with interparietal split and one fetus with fused distal cartilages of ribs.
These findings were not considered to be test item-related as they were at an isolated incidence (the three malformations) and/or were noted in a similar incidence than in controls and/or HCD (interparietal split and fused cartilage of ribs).
There were no skeletal malformations at 60 mg/kg/day.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The only soft tissue malformation was dilated pulmonary trunk in one fetus at 500 mg/kg/day, which was not considered to be test item-related (isolated incidence).

Other effects:

effects observed, treatment-related

Description (incidence and severity):

External variations
The only external variation noted in test item groups was limb hyperflexion in one fetus at 500 mg/kg/day and was not considered to be test item-related (isolated fetus and incidence comparable to historical control data).

Cartilages
Cartilages of short, unossified or incomplete/bipartite/dumbbell ossification of bones were present, except for the short rib in one fetus of the 500 mg/kg/day group. In view of the isolated incidence, this finding was considered incidental.
Besides, one fetus at 170 mg/kg/day had fused distal cartilages of ribs which was discussed below (malformation).

Skeletal Variations
At 500 mg/kg/day and when compared with controls, there were higher fetal (69.6% vs. 41.3%) and litter (95.7% vs. 79.2%) incidences of fetal skeletal variations as showed by the statistically significant mean percentage of affected fetuses per litter (71.2% vs. 41.3%, p < 0.01). This test item effect was considered to be non-adverse (variations) and to represent ossification delays (the most affected bones being supraoccipital, caudal vertebrae, proximal phalanx of forepaws, metacarpal, 1st metatarsal and distal phalanx of hindpaws).
At 60 and 170 mg/kg/day, litter incidences of fetal skeletal variations were also higher than in controls (87 and 91.7% vs. 79.2%, respectively) but fetal incidences (43.6 and 48% vs. 41.3%, respectively) and mean percentages of affected fetuses per litter (44.9 and 47.6 vs. 41.3, respectively) remained comparable with controls. The most relevant variation was unossified proximal phalanx of forepaws at 170 mg/kg/day.
These findings were non-adverse and considered of no biological significance in absence of effect on mean fetal body weight.

Visceral Variations (See table 9).
None of the soft tissue variations were considered to be related to the test item treatment: they were not dose-related, were noted in an isolated fetus, and/or had incidences lower than in HCD and/or controls.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

170 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Fetal abnormalities

Abnormalities:

effects observed, non-treatment-related

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

  Table 1: Clinical signs

Dose-level (mg/kg/day)	0	60	170	500
Ptyalism		23	24	23^a
Piloerection				2^a
Round back				1^a
Emaciated appearance				2^a
Soft feces				1^a
Soiled urogenital area				1^a
Abdominal breathing				1
Loud breathing			1	1
Dacryhorrhea				1
Chromorhinorrhea				1
Area of hair loss on limbs	2		1
Number of affected animals	2/24	23/24	24/24	23/23

^a: including female H21169.

Table 2: Body weight and body weight change

 

Dose-level (mg/kg/day)	0	60	170	500
Body weight
Day 6p.c.	280	290	286	287
		+4	+2	+3
Day 9p.c.	286	297	290	273
		+4	+1	-5
Day 12p.c.	306	314	310	289*
		+3	+1	-6
Day 15p.c.	326	337	330	304**
		+3	+1	-7
Day 18p.c.	369	385	376	337**
		+4	+2	-9
Day 21p.c.	414	434	421	369#
		+5	+2	-11
Body weight change
Days 6 - 9p.c.	+6	+7	+4	-13#
Days 9 - 12p.c.	+20	+17	+20	+16
Days 12 - 15p.c.	+20	+22	+20	+15
Days 15 - 18p.c.	+43	+48	+46	+34*
Days 18 - 21p.c.	+45	+49	+45	+27#
Days 6 - 21p.c.	+134	+144	+135	+82#

Statistical significance: *:p < 0.05; **:        p < 0.01; #:         p < 0.001.

Initalic: % from controls.

Table 3: Food consumption

 

Dose-level (mg/kg/day)	0	60	170	500
Days 6 - 9p.c.	17	19	16	10#
		+12	-6	-41
Days 9 - 12p.c.	22	22	23	16#
		0	+5	-27
Days 12 - 15p.c.	25	26	26	20#
		+4	+4	-20
Days 15 - 18p.c.	28	29	28	22#
		+4	0	-21
Days 18 - 21p.c.	28	30	29	22**
		+7	+4	-21

Statistical significance:**:              p < 0.01; #:         p < 0.001.

Initalic: % from controls.

Table 4: Maternal necropsy findings

 

Dose-level (mg/kg/day)	0	60	170	500
Colored content in uterine horn	1
Deformation of spleen (bilobed)		1
Dilated renal pelvis		1
Whitish area on forestomach				1
Placentas fused	1
Number of affected animals	2/24	2/23	0/24	1/23

 

Table 5: Net body weight change

 

Dose-level (mg/kg/day)	0	60	170	500
Gravid uterus weight^a	96.0	102	96.5	82.5*
		+6	+1	-14
Carcass weight^a	318	332	325	286#
		+4	+2	-10
Net body weight change from Day 6p.c.	+38.0	+41.3	+38.8	-0.3#

Statistical significance: *:p < 0.05; #:         p < 0.001.

Initalic: % from controls.

^a: rounded to three significant digits when applicable.

Table 6: Hysterectomy

 

Dose-level (mg/kg/day)	0	60	170	500
Number of females with live fetuses	24	23	24	23
Number ofcorpora lutea	14.7	14.3	14.3	15.1
Number of implantation sites	12.8	13.2	12.7	13.6
Pre-implantation loss (%)	11.8	7.7	10.3	9.6
Number of fetuses	12.0	12.7	11.8	12.4
Dead fetuses (%)	0.0	0.0	0.0	0.3
Number of early resorptions	0.6	0.5	0.7	1.1
Number of late resorptions	0.2	0.1	0.2	0.0
Post-implantation loss (%)	6.5	4.2	6.9	8.5

Table 7: Fetal body weight

 

Dose-level (mg/kg/day)	0	60	170	500
Mean fetal body weight (g)	5.68	5.73	5.76	4.61#
		+1	+1	-19

Statistical significance: #:p < 0.001.

Initalic: % from controls.

 

Table 8: Exernal malformations

 

Dose-level (mg/kg/day)	0	60	170	500	HCD
Number of litters	24	23	24	23	388
Number of fetuses	288	291	284	284	5000
Ombilical hernia, F (L)		0.3 (4.3)			0.4 (4.8)
Anal atresia, F (L)			0.4 (4.2)^a		0.4 (5.0)
Thread-like tail, F (L)			0.4 (4.2)^a		-
Litters with external malformation, n (%)	0 (0.0)	1 (4.3)	1 (4.2)	0 (0.0)	11 (2.8)
Fetuses with external malformation, n (%)	0 (0.0)	1 (0.3)	1 (0.4)	0 (0.0)	13 (0.3)

F: fetal incidence; L:          litter incidence.

HCD: Historical control data (Sprague-Dawley rats from the Supplier Janvier; signed in December 2016): max fetal incidence       (max litter incidence) for the findings.

-: none in HCD.

^a: fetus H21140-13.

Table 9: Soft issue malformations

 

Dose-level (mg/kg/day)	0	60	170	500	HCD
Number of litters	24	23	24	22	387
Number of fetuses	138	142	136	136	2404
Small spleen, F (L)				0.7 (4.5)	-
Dilated renal pelvis, F (L)	2.9 (12.5)	5.6 (21.7)	1.5 (8.3)		9.5 (28.6)
Short innominate artery, F (L)		0.7 (4.3)	2.2 (12.5)	0.7 (4.5)	3.7 (22.7)
Absent innominate artery, F (L)	1.4 (8.3)	2.8 (17.4)	1.5 (8.3)	1.5 (4.5)	5.1 (25.0)
Dilated ureter, F (L)	2.2 (12.5)	2.1 (13.0)	0.7 (4.2)	0.7 (4.5)	7.1 (28.0)
Thymus: reddish focis, F (L)		1.4 (8.7)			-
Litters with soft tissue variation, n (%)	4 (16.7)	10 (43.5)	7 (29.2)	4 (18.2)	86 (22.2)
Fetuses with soft tissue variation, n (%)	6 (4.3)	16*(11.3)	7 (5.1)	5 (3.7)	119 (5.0)

F: fetal incidence; L:         litter incidence.

Statistical significance: *: p < 0.05.

HCD: Historical control data (Sprague-Dawley rats from the Supplier Janvier; signed in December 2016): max fetal incidence       (max litter incidence) for the findings.

-: none in HCD.

 

Applicant's summary and conclusion

Conclusions:

The test item was administered by gavage, once daily, from Days 6 to 20 p.c. inclusive, to pregnant Sprague-Dawley rats at dosages of 60, 170 and 500 mg/kg/day.

At 500 mg/kg/day, maternal toxicity (poor health condition, absence of food intake and body weight loss) found in a few females resulted in one death on Day 19 p.c. Otherwise, ptyalism was the main clinical sign in this group. There was also low mean food consumption throughout the treatment period associated with low mean body weight. These effects correlated with the low mean carcass weight and mean net body weight change from Day 6 p.c. at necropsy. There were also associated low mean gravid uterus weight, low mean fetal weight and fetal ossification delays.

At 60 and 170 mg/kg/day, there were no adverse findings in dams and litters.

Under the experimental conditions and results of this study:
- the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 170 mg/kg/day in view of poor general condition, death and effect on body weight and food consumption at 500 mg/kg/day,
- the NOAEL for embryo-fetal development was considered to be 170 mg/kg/day based on decreased mean fetal weight at 500 mg/kg/day.

Executive summary:

The objective of this GLP study was to evaluate the potential toxic effects of the test item on the pregnant female and on embryonic and fetal development, following daily oral administration (gavage) to pregnant female rats from implantation to the day before scheduled hysterectomy [Days 6 to 20 post-coitum (p.c.) inclusive].

 

Methods

Three groups of 24 time-mated female Sprague-Dawley rats received the test item at 60, 170 or 500 mg/kg/day by oral route (gavage) once daily from Days 6 to 20 p.c. A constant dosage-volume of 5 mL/kg/day was used. Another group of 24 rats received the vehicle alone (corn oil) under the same experimental conditions, and acted as a control group.

 

Formulation concentrations were checked in the first and last weeks of treatment in the study.

 

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded every 2 to 3 days. On Day 21 p.c., females were sacrificed and submitted to a macroscopic post-mortem examination of principal thoracic and abdominal organs. Hysterectomy was performed and the numbers of corpora lutea, implantations, uterine scars, early and late resorptions, live and dead fetuses were recorded. The fetuses were sexed, weighed and examined for external, soft tissues and skeletal (cartilages + bones) abnormalities.

 

Results

The test item concentrations in the dose formulations analyzed were within an acceptable range of variations when compared with the nominal values (± 15% required). No test item was observed in the control dose formulations.

 

On Day 21 p.c., all surviving females were pregnant and had live fetuses, except one non-pregnant female at 60 mg/kg/day.

One female treated at 500 mg/kg/day was found dead on Day 19 p.c. following poor health condition (no food consumption, body weight loss, clinical signs as breathing difficulties, emaciation, round back). The cause of the death was not determined but considered to be test item-related, because there were two other females in the same group with comparable adverse findings towards the end of the study.

Otherwise, the main test item-related clinical sign was ptyalism noted in almost all test item-treated females. Besides, a test item effect was not ruled out for loud breathing noted in one female at 170 mg/kg/day.

 

At 500 mg/kg/day, there was a lower mean food consumption throughout the treatment period (down to -41% vs. controls on Days 6 to 9p.c., around -21% by the end of the study) correlating with mean body weight loss from Days 6 to 9 p.c. followed with low mean body weight gains which resulted in low mean body weight (down to -11% vs. controls on Day 21 p.c., p < 0.001).

There were no effects on mean food consumption, mean body weight and mean body weight change at 60 and 170 mg/kg/day.

There were no test item-related necropsy findings in surviving females. At 500 mg/kg/day, correlating with the effects on mean body weight, there were lower mean carcass weight (-10% p < 0.001 vs. controls) and mean net body weight change from Day 6 p.c. (-0.3 g vs. +38 g p < 0.001 vs. controls). There were no effects on mean carcass weight and on mean net body weight change from Day 6 p.c. at 60 and 170 mg/kg/day.

There were no effects on mean hysterectomy data and sex ratio. At 500 mg/kg/day, there was a low mean gravid uterus weight (-14% p < 0.05, vs. controls) correlating with lower mean fetal body weight (-19% vs. controls, adverse).There were no effects on mean gravid uterus weight and mean fetal body weight at 60 and 170 mg/kg/day.

 

At fetal examination, there were no test item treatment-related external or soft tissue variations and malformations, or skeletal malformations. At 500 mg/kg/day, there were higher fetal (69.6% vs. 41.3% in controls) and litter (95.7% vs. 79.2%) incidences of fetal skeletal variations (non-adverse) representing ossification delays. At 60 and 170 mg/kg/day, the slightly higher litter incidences of fetal skeletal variations (respectively 87 and 91.7% vs. 79.2% in controls) were considered of no biological significance.

 

Conclusion

The test item was administered by gavage, once daily, from Days 6 to 20 p.c. inclusive, to pregnant Sprague-Dawley rats at dosages of 60, 170 and 500 mg/kg/day.

 

At 500 mg/kg/day, maternal toxicity (poor health condition, absence of food intake and body weight loss) found in a few females resulted in one death on Day 19 p.c. Otherwise, ptyalism was the main clinical sign in this group. There was also low mean food consumption throughout the treatment period associated with low mean body weight. These effects correlated with the low mean carcass weight and mean net body weight change from Day 6 p.c. at necropsy. There were also associated low mean gravid uterus weight, low mean fetal weight and fetal ossification delays.

At 60 and 170 mg/kg/day, there were no adverse findings in dams and litters.

 

Under the experimental conditions and results of this study:

.         the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 170 mg/kg/day in view of poor general condition, death and effect on body weight and food consumption at 500 mg/kg/day,

.         the NOAEL for embryo-fetal development was considered to be 170 mg/kg/day based on decreased mean fetal weight at 500 mg/kg/day.