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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 March 2016 - 05 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 January 2001
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, le Genest-Saint-Isle, France
- Sex: female
- Age at study initiation: at the beginning of the treatment period, the animals were 10-11 weeks old
- Mean body weight at study initiation: mean body weight of 296 g (range: 251 g to 346 g)
- Housing: the animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing autoclaved sawdust
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 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: 09 May 2016 to 05 July 2016.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Suspension in the vehicle.

Preparation procedure:
According to a homogeneity / stability describing the preparation procedure for a range of concentrations covering the lowest and highest used in this study.

VEHICLE
- Justification for use and choice of vehicle: suitable formulation in corn oil
- Concentration in vehicle: 10, 50 and 200 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: HPLC-UV (High Performance Liquid Chromatography with UV detection).
Once in Weeks 1, 2 and 3 of treatment in the study.
A sample was taken from control and test item dose formulations and analysed using the validated method.
Duration of treatment / exposure:
Day 6 to Day 20 p.c.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
26 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected by the Sponsor based on a preliminary study for effects on embryo-fetal development by the oral route in rats.

In this study, three groups of eight pregnant female Sprague-Dawley rats received the test item, daily by oral administration (gavage) throughout gestation (Day 6 to 20 p.c.) at dose levels of 100, 300 or 1000 mg/kg/day. An additional group of 8 pregnant females received the vehicle control, corn oil, under the same experimental conditions. The dosing volume was 5 mL/kg/day.
The dose-level of 1000 mg/kg/day was associated with ptyalism in all animals and moderate effects (i.e. lower body weight gain together with lower food consumption at start of the study followed with higher body weight gain). Minimal increases in the absolute and relative liver weights were observed.
The dose-level of 300 mg/kg/day was associated with ptyalism in one female.
The dose-level of 100 mg/kg/day was not associated with any test item effects.
Therefore, 1000 mg/kg/day were selected as the high dose-level. The low-dose and mid-dose were selected using a ratio representing approximately a 4- to 5-fold interval (i.e. 50 and 250 mg/kg/day).

- Rationale for animal assignment: computerized randomization procedure

Examinations

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

CLINICAL OBSERVATIONS:
- 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.
- Organs examined: principal thoracic and abdominal organs. Kidneys and liver from all study animals were weighed.

MICROSCOPIC EXAMINATION:
A microscopic examination was performed on:
- all macroscopic lesions from all control and test item-dose animals (groups 1 to 4) sacrificed at the end of the treatment period and from all animals that died,
- corresponding tissues of five controls.
Ovaries and uterine content:
The ovaries and uterine content were examined after termination: Yes
Examinations included:
- Gravid uterus weight,
- Number of corpora lutea,
- Number of implantations sites,
- Number of early resorptions,
- Number of late resorptions,
- Number of dead and live fetuses,
- Number of uterine scars.
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 dead and live, 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) / Number of corpora lutea
% Post-implantation loss = 100 * (Number of implantation sites - Number of live fetuses) / Number of implantation sites

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See table 1.
Test item treatment-related clinical signs of minor toxicological importance consisted of:
- tremors and hypoactivity noted on one occasion and followed by piloerection for 3 days in 1/24 females given 1000 mg/kg/day,
- ptyalism observed in a few females given 50 or 250 mg/kg/day and in most of the surviving females given 1000 mg/kg/day for a 3- to 15-day duration.

Reddish/brownish vaginal discharge was transiently observed in isolated females given 0, 50 or 1000 mg/kg/day. Cutaneous lesions and scabs on the back were observed in 1/25 females given 50 mg/kg/day. These findings were considered to be unrelated to the test item treatment, as they are commonly observed in rats of this strain and age.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no test item treatment-related unscheduled deaths in any of the groups.

In all groups (including control), there were found dead animals. At histopatholical examination, the cause of death was considered to be related to technical issues consecutive to the administration of oily formulations during the gavage procedure:
- at 1000 mg/kg/day, one female died after treatment on Day 8 p.c. and one female was found dead on Day 16 p.c. Prior to death, both females showed piloerection and ptyalism, and one female showed emaciated appearance from Day 8 p.c.. A very slight body weight loss (-1 g) was recorded in one female between Days 12 and 15 p.c. together with slightly reduced food consumption. These females were pregnant,
- at 250 mg/kg/day, one female was found dead on Day 10 p.c. without relevant clinical signs prior to death. This female was pregnant,
- at 50 mg/kg/day, one female was found dead on Day 18 p.c. without relevant clinical signs prior to death. At histopathological examination, the cause of death was considered to be technical issues at the gavage procedure. This female was pregnant,
- at 0 mg/kg/day, one female was found dead on Day 17 p.c. without relevant clinical signs prior to death. A very slight body weight loss (-1 g) was observed over the periods of Days 6-9 and 12-15 p.c.. At histopathological examination, the cause of death was considered to be technical issues at the gavage procedure. This female was pregnant.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no effects on mean body weight or mean body weight change at any dose-level.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg/day, when compared to controls, a lower mean food consumption (-24%, p < 0.001) was recorded between Days 6 and 9 p.c., with a return to control values thereafter and therefore considered to be non-adverse.

At 50 and 250 mg/kg/day there were no effects of treatment with the test item.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See table 2.
When compared to controls, the mean absolute and relative liver weights were higher (statistically significant) in females treated at 1000 mg/kg/day (p < 0.05 for the absolute weight; p < 0.01 for the relative weight).
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Unscheduled deaths
- Control animal: one female found dead on Day 17 p.c.
Translucent content was observed in the thoracic cavity. Despite the absence of histopathological examination, this observation was considered to be a sequela consecutive to a mistake in the technical gavage procedures.

- Low-dose group (50 mg/kg/day): one female found dead on Day 18 p.c.
Translucent content was observed in the thoracic cavity and in the pericardium along with red thick content. Despite the absence of histopathological examination, these observations were considered to be sequelae consecutive to a mistake in the technical gavage procedures.

- Mid-dose group (250 mg/kg/day): one female found dead on Day 10 p.c.
Red content was seen in the thoracic cavity and in the pericardium (noted thick) along with translucent liquid content. Despite the absence of histopathological examination, these observations were considered to be sequelae consecutive to a mistake in the technical gavage procedures.

- High-dose group (1000 mg/kg/day): two females found dead on Days 16 or 8 p.c., respectively.
Red liquid content was seen in the thoracic cavity of these both animals along with red or translucent content in the pericardium.
In addition, several changes were observed in one female. These included perforation of the esophagus, white discoloration and edema of the thymus, foamy content in the lungs, and red content in the stomach, duodenum and ileum.

A histopathological examination was performed on the esophagus, heart, thymus and lungs of one female.
Rupture of the esophagus was observed, along with acute inflammation in the adjacent tissues (with hemorrhage and eosinophilic material consistent with fibrin and foreign bodies considered to be food debris, and bacteria).
There was a moderate acute inflammation of the epicardium/pericardium with prominent edema, eosinophilic material (consistent with fibrin), hemorrhage, and numerous clusters of bacteria that correlated with the translucent content seen at necropsy.
Minimal lymphoid atrophy along with a moderately increased number of apoptotic cells was seen in the thymus. In addition, the tissues adjacent to the thymus showed inflammation along with edema and eosinophilic material (consistent with fibrin), which correlated with white discoloration and edema seen at macroscopic examination.
Minimal acute inflammation of the pleura was seen in the lungs, along with slight congestion. There were, however, no histopathological correlates for the foamy content observed at necropsy.

Again all these changes were considered to be consequences of a mistake in the technical gavage procedures.

Terminal sacrifice
- Control animals
Thick white content was observed in the thoracic cavity of two females. This was accompanied by enlargement of the mediastinal/thoracic lymph nodes in one female or irregular discoloration of the liver in one female.

From a histological viewpoint, the mediastinal/thoracic lymph nodes of one female showed a moderately increased number of plasma cells, which correlated with the enlargement noted at gross examination and minimal inflammation of the adjacent tissues that looked to be consequences of a mistake in the technical gavage procedures.

No relevant changes were observed at macroscopic/microscopic examinations of the trachea, esophagus, lungs, heart, thymus or placenta from two controls, which were examined.

- Low-dose group (50 mg/kg/day)
Thick content was observed in the thoracic cavity of two females. This was accompanied by enlargement of the mediastinal/thoracic lymph nodes in one animal.

At histological examination, the mediastinal/thoracic lymph nodes of one female showed a moderately increased number of plasma cells, which correlated with the enlargement noted at gross examination and minimal inflammation of the adjacent tissues that looked to be consequences of a mistake in the technical gavage procedures.

- Mid-dose group (250 mg/kg/day)
A white mass was observed in the esophagus of one female.
Thick white content was observed in the pericardium of two females, along with oily translucent content (in one female) or thickening of the pericardium (in one female).
Other changes included enlargement of the mediastinal/thoracic lymph nodes (in one female), reduced sized with red discoloration of the thymus, and irregular coloration of the lungs (in one female).

Moderate to marked inflammation of the epicardium/pericardium along with hemorrhage and eosinophilic material in the pericardium (consistent with fibrin) correlated with observations at gross examination of the heart of both the above animals. In one animal, clusters of bacteria were present.

In one female, there was an abscess (graded moderate) in the tissues adjacent to the esophagus that correlated with the mass seen at necropsy. Foreign bodies consistent with food debris and bacteria were seen in this abscess. The mediastinal/thoracic lymph nodes of this animal showed a moderately increased number of plasma cells, which correlated with the enlargement noted at gross examination, and slight inflammation of the adjacent tissues. These changes were considered to be the consequences of a mistake in the technical gavage procedures.

In one female, lymphoid atrophy, graded moderate, along with hemorrhage and inflammation in the adjacent tissues was seen in the thymus, correlating with changes seen at macroscopy.

No relevant changes were seen in the lungs. The irregular coloration noted at gross examination correlated with minimal congestion.

- High-dose group (1000 mg/kg/day)
A red irregular coloration of the lungs was seen in one female, correlating with slight congestion at histology. This observation was considered to be non-specific and probably agonal.

Enlarged placenta was seen in one female, correlating at histology with dilation of vessels and enlarged cells in the basal zone. It was considered likely that this observation in an isolated animal was incidental.

Discussion
All changes, except foamy content or irregular color seen in lungs, were consistent with more or less developed lesions secondary to trauma induced by the technical gavage procedures.
The changes were seen in decedent animals or in those sacrificed at the end of the study and included material in the thoracic cavity (recorded at necropsy as white, red or translucent content) and inflammation seen microscopically in the heart (epicardium/pericardium) or tissues adjacent to the organs of the thoracic cavity (e.g thymus, mediastinal/thoracic lymph nodes, lungs). They were considered to be secondary to perforation of the esophagus during the gavage procedure as they were seen macroscopically and microscopically in one female or suggested in one female where an abscess with foreign bodies was seen close to the esophagus.

Any relationship of all these lesions with the test item was excluded.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Slightly higher mean post-implantation loss was noted in females given 50, 250 or 1000 mg/kg/day, when compared with controls. As these differences were not dose-related, within or close to the limits of the historical control data and did not impact the mean number of live fetuses, a test item treatment relationship was considered unlikely.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
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

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
See table 3.
Mean fetal body weight was lower (-8% vs. controls, p < 0.001) in the 1000 mg/kg/day group.
As the difference was minimal and close to the lower limit of the Historical Control Data, it was considered to be of minor toxicological importance.

There were no effects on mean fetal body weight in the 50 and 250 mg/kg/day groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex-ratio (percentage of male fetuses) was unaffected by the test item treatment at any dose-level.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Variations
There was no test item treatment-related increase of the frequencies of external variations.
In the 1000 mg/kg/day group, one fetus had a curled tail. This is a common finding in this species and strain, therefore any relationship to the test item treatment was considered unlikely.

Malformations
There was no test-item treatment related external malformations.
In each mid- and high-dose group, one fetus had external malformations commonly observed in this species and strain (Morita et al., 1987):
- 250 mg/kg/day: one fetus (fetal body weight: 2.79 g): agnathia and gastroschisis [absent hyoid and mandible, split palate, absent thoracic hemivertebra(e) and hemicentrum, fused sternebra(e), and absent or fused ribs at skeletal examination],
- 1000 mg/kg/day: one fetus (fetal body weight: 6.15 g): anal atresia and acaudia [absent thoracic, lumbar, sacral and caudal vertebra(e), and fused centrum of sacral vertebra(e) at skeletal examination].
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
See table 4.
Cartilage
At 1000 mg/kg/day and when compared with controls, there was an increase in ossification delay (cartilage of metatarsal bone present but not ossified) which was consistent with the slightly lower mean fetal body weight recorded at this dose-level. Although the incidences were below the maximum values of the Historical Control Data, this finding was considered to be associated to the test item treatment but of minor toxicological importance.

Variations
At 1000 mg/kg/day and when compared with controls, there was an increase in ossification delay (unossified metatarsal bone) which was consistent with the slightly lower mean fetal body weight recorded at this dose-level. Although the incidences were below the maximum values of the Historical Control Data, this finding was considered to be associated to the test item treatment but of minor toxicological importance.

Malformations
In the 1000 mg/kg/day group, two litters had one or two malformed fetuses: one with cervical rib, another with absent thoracic and lumbar vertebra(e) and branched rib(s), and another one with absent lumbar, sacral and caudal vertebra(e) and fused centrum of sacral vertebra(e).

In the 250 mg/kg/day group, one litter had a malformed fetus with absent hyoid and mandible, split palate, fused sternebrae, absent thoracic hemivertebra(e) and hemicentrum and absent or fused rib(s).

In the 50 mg/kg/day group, no malformed fetuses were observed.

In the control group, one litter contained one malformed fetus with less than 15 caudal vertebrae.

Similar findings (axial skeleton defects) were observed in control, mid- and high-dose groups. Visceral abnormalities were recorded in the low-dose group, only. Head and wall (midline) defects were recorded in the mid-dose group, only. There was no obvious relationship between these findings and therefore, any link to a test item treatment was considered to be unlikely.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Variations
When compared with controls, there was a tendency towards increased fetal and litter incidences of absent innominate artery at all dose-levels (from 2.8% to 3.6% and from 12.0% to 18.2%, fetal and litter incidences, respectively). Taking into account that incidences (fetal and litter) of this non-adverse finding were lower than the maximal incidence of the Historical Control Data, a test item treatment-related effect was considered to be unlikely.

Other soft tissue variations (i.e. dilated renal pelvis, dilated ureter, reddish foci on thymus and short innominate artery) observed at comparable incidence in control and test item-treated groups were considered to be unrelated to the test item treatment.

Malformations
There was no test item treatment-related increase in the frequencies of soft tissue malformations.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
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

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Clinical signs

 

Dose-level (mg/kg/day)

0

50

250

1000

Piloerection

 

 

 

1

(from Day 8 to
Day 10p.c.)

Tremors and hypoactivity

 

 

 

1

(Day 7p.c.)

Ptyalism

 

2

(Day 16p.c. or from Day 9 to Day 10p.c.)

2(Day 6p.c. or from Day 6 to Day 17p.c.)

22

(usually from the first or second week of treatment)

Reddish/brownish vaginal discharge

1

(from Day 17 to 18p.c.)

2

(from Day 15 to Day 16p.c. or from Day 20 to Day 21p.c.)

 

3

(Day 14p.c.or from Day 14 to Day 17p.c.)

Cutaneous lesions/scabs on back

 

1

(from Day 17 to Day 21p.c.)

 

 

Number of affected animals

1/25

5/25

2/25

22/24

( )     : in brackets: daysp.c.of occurrences.

Table 2: Organ weights

 

Sex

Female

Group

2

3

4

Dose-level (mg/kg/day)

50

250

1000

Number of animals

25

25

24

- Final body weight

0

-1

-2

- Liver

. absolute

-2

-2

+10*

. relative

-1

-1

+11**

Statistically significant from controls:

 *      : p < 0.05.

**     : p < 0.01.

The significance concerned the organ weight values and not the percentages.

Table 3: Fetal body weight changes

 

Dose-level (mg/kg/day)

0

50

250

1000

HCD

Mean fetal body weight (g)

5.88
-

5.81
(-1)

5.74
(-2)

5.40#
(-8)

[5.5; 5.9]

Mean fetal body weight
of males (g)

6.06
-

5.97
(-1)

5.87
(-3)

5.55#
(-8)

[5.7; 6.1]

Mean fetal body weight
of females (g)

5.69
-

5.63
(-1)

5.58
(-2)

5.23#
(-8)

[5.4; 5.7]

Mean percentage
of male fetuses (%)

54.0

51.8

53.8

53.7

[44.0; 55.4]

( )     : in brackets, percentage differencevs.controls.

HCD      : Historical Control Data (control data collected from nine studies covering a period ranging
          from February 2014 to July 2016), [min.; max.].

Statistically significant#: p < 0.001.

-       : not applicable.

Table 4: Skeletal examinations

Cartilage

Fetal (Litter) incidences (%) of cartilage findings

 

Dose-level (mg/kg/day)

0

50

250

1000

HCD

Dams with live fetuses

25

25

24

22

388

Number of live fetuses

169

158

146

150

2596

Litters affected, n (%)

24 (96.0)

24 (96.0)

22 (91.7)

21 (95.5)

236 (60.8)(b)

Fetuses affected, n (%)

110 (65.1)

115 (72.8)

98 (67.1)

80 (53.3)

990 (38.1)(b)

Cartilage of metatarsal
bone present, F(L)

8.3 (36.0)

10.8 (56.0)

14.4 (45.8)

22.0#(63.6)

36.8 (72.7)(a)

F      : fetal incidence.

L      : litter incidence.

Statistically significant#: p < 0.001 for the number of fetuses affected.

HCD      : Historical Control Data (control data collected from nine studies covering a period ranging
         from February 2014 to July 2016).

(a)     : maximum incidence.

(b)     : mean incidence.

Variations

Fetal (Litter) incidences (%) ofskeletal variations

 

Dose-level (mg/kg/day)

0

50

250

1000

HCD

Dams with live fetuses

25

25

24

22

388

Number of live fetuses

169

158

146

150

2596

Litters affected, n (%)

24 (96.0)

24 (96.0)

22 (91.7)

21 (95.5)

345 (88.9)(b)

Fetuses affected, n (%)

111(65.7)

115 (72.8)

99 (67.8)

83 (55.3)

1267 (48.8)(b)

Metatarsal bone: unossified

first metatarsal, F(L)

8.3 (36.0)

10.8(56.0)

14.4(45.8)

22.0#(63.6)

36.8 (72.7)(a)

F      : fetal incidence.

L      : litter incidence.

Statistically significant#: p < 0.001 for the number of fetuses affected.

HCD      : Historical Control Data (control data collected from nine studies covering a period ranging
         from February 2014 to July 2016).

(a)     : maximum incidence.

(b)     : mean incidence.

Applicant's summary and conclusion

Conclusions:
The test item was administered by gavage, once daily, from Day 6 to Day 20 p.c., inclusive, to pregnant Sprague-Dawley rats at the dose-levels of 50, 250 and 1000 mg/kg/day. The control group received the vehicle, corn oil, under the same experimental conditions.
On the basis of the results obtained in this study:
- the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 1000 mg/kg/day,
- the NOAEL for embryo-fetal development was considered to be 1000 mg/kg/day.
Executive summary:

The potential toxic effects of m/p-diol on the pregnant female and on embryonic and fetal development was evaluated following daily oral administration (gavage) to pregnant female ratsfrom implantation until the day before scheduled hysterectomy [Day 6 to Day 20 post-coitum (p.c.) inclusive]. This GLP study was carried out according to OECD test guideline No. 414 (22 January 2001). Three groups of 26 time-mated female Sprague-Dawley rats received m/p-diol Day 6 to Day 20 p.c. by the oral route (gavage), at a dose-level of 50, 250 or 1000 mg/kg/day, once daily from Day 6 to Day 20 p.c. inclusive. Another group of 26 time-mated rats received the vehicle only, corn oil, under the same experimental conditions and acted as a control group. A constant dosage volume of 5 mL/kg/day was used. The test item concentrations in the dose formulations were determined. The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 21 p.c., the females were sacrificed and a macroscopic post-mortem examination was performed. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, uterine scars, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and skeletal abnormalities (including cartilage). Samples of the kidneys and the liver of all dams were collected, weighed and preserved in 10% buffered formalin. A microscopic examination was performed on all macroscopic lesions.

The concentrations of the test item in the dose formulations (-7.7% to -1.1%) remained within the acceptable range of variations (± 15%)when compared to the nominal concentrations. There were 25, 25, 24 and 22 females with live fetuses in the groups given 0, 50, 250 and 1000 mg/kg/day, respectively. There were no test item treatment-related unscheduled deaths in any group. Tremors, hypoactivity and piloerection were transiently observed in 1/24 dams given 1000 mg/kg/day. Ptyalism was noted in 2/25 females given 50 or 250 mg/kg/day and in 22/24 dams given 1000 mg/kg/day. There were no effects on body weight or body weight change at any dose-level. At 1000 mg/kg/day, lower food consumption (-24% vs. controls, p < 0.001) was observed between Days 6 to 9 p.c., with a return to control values thereafter. At 1000 mg/kg/day, non-adverse, statistically significantly, higher absolute and relative liver weights (+10% and +11% vs. controls, respectively) were noted.

There were no test item treatment-related macroscopic or microscopic lesions at any dose-level. There were no test item-related effects at any dose-level. There were no effects on gestation parameters at any dose-level. At 1000 mg/kg/day and when compared with controls, the lower mean fetal body weights observed in the high-dose group were statistically significant (p < 0.001). This finding, which was considered to be of minor toxicological importance, was close to the lower limit of the historical control data. The sex ratio was unaffected by the test item treatment at any dose-level. There were no test item treatment-related variations at external/oral cavity examination of the fetuses. There were no test item-related variations and malformations at soft tissue examination. At 1000 mg/kg/day, when compared with controls, there were higher litter and fetal incidences of fetuses with unossified first metatarsal. The incidences being lower than maximum means of historical control data, these effects were considered non adverse.

There were no test item-related malformations at skeletal examination.

The test item was administered by gavage, once daily, from Day 6 to Day 20 p.c., inclusive, to pregnant Sprague-Dawley rats at the dose-levels of 50, 250 and 1000 mg/kg/day. The control group received the vehicle, corn oil, under the same experimental conditions.

 On the basis of the results obtained in this study:

.         the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 1000 mg/kg/day,

.         the NOAEL for embryo-fetal development was considered to be 1000 mg/kg/day.