Amides, C12-18, N-[3-(dimethylamino)propyl]

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 September 2016 - 29 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

January 2001

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd 3 november 2015

Limit test:

no

Test material

Test material information

1

Reference substance name:

N-[3-(dimethylamino)propyl] C 12-C18 alkylamide

EC Number:

932-121-8

Cas Number:

1147459-12-8

Molecular formula:

UVCB substance not applicable

IUPAC Name:

N-[3-(dimethylamino)propyl] C 12-C18 alkylamide

Test material form:

other: Paste

Details on test material:

- State of aggregation: paste
- Density: 0.885 g/cm3 at 20°C (solid)
- Analytical purity: 100% (UVCB)
- Color gardner 2: 7
- Purity test date: 18 May 2016
- Lot/batch No.: BS-160515001-001
- Expiration date of the lot/batch: 15 May 2021
- Storage condition of test material: At room temperature container flushed with nitrogen
- Solubility and stability of the test substance in the solvent/vehicle: stable as solution in corn oil for at least 5 hours at room temperature and 11 days in the refrigerator (confirmed over the concentration range 1 to 200 mg/mL)
OTHER SPECIFICS: no correction factor applied for purity; pH 11 at 1%.
Mean mol weight ~ 296 Range 228- 368

batch BS-160515001-001

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature container flushed with nitrogen
- Solubility and stability of the test substance in the solvent/vehicle: stable as solution in corn oil for at least 5 hours at room temperature and 11 days in the refrigerator (confirmed over the concentration range 1 to 200 mg/mL)

Test animals

Species:

rat

Strain:

other: Crl:WI (Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-14 weeks.
- Weight at study initiation: 174-231 g
- Fasting period before study: none
- Housing: individually in Macrolon plastic cages (MIII type, height 18 cm). Sterilized sawdust as bedding material and paper as cage enrichment/nesting material were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water
- Acclimation period: At least 5 days prior to treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7 - 21.2
- Humidity (%): 50.1 – 74.8
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 06 September 2016 (first delivery of mated females) To: 29 September 2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test item (0.885 g/cm3 at 20 °C) and vehicle (0.92 g/cm3). No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch
- Amount of vehicle (if gavage): 5 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (12 September 2016), according to a validated method (LC-MS/MS, validated in a range 1-250 mg/g). Samples of
formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

Details on mating procedure:

Untreated females were mated at the Supplier, and were at Day 0 or 1 post-coitum on arrival at the Test Facility.
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

From Days 6 to 20 post-coitum, inclusive

Frequency of treatment:

Once daily, 7 days/week

Duration of test:

14 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

concurrent vehicle controls

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

40 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

100 mg/kg bw/day

No. of animals per sex per dose:

22 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a 28-day repeated dose study (OECD 407), with a 7-day dose range finder in advance, and a combined 28-day repro screening study (OECD 422), with a 14-day dose range finder in advance.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for mortality and viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily for clinical signs from day 2 post-coitum onwards up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum

WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced, as no treatment related effect was suspected

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: ovaries and uterine horns

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter ]
- Head examinations: Yes: [half per litter]

Statistics:

The following statistical methods were used to analyze the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.
• The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.
• Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

For each litter the following calculations were performed:
Pre-implantation loss (%) = ((number of corpora lutea - number of implantation sites)/number of corpora lutea) x 100%
Post-implantation loss (%) = ((number of implantation sites - number of live fetuses)/number of implantation sites) x 100%
Viable fetuses affected/litter (%) = ((number of viable fetuses affected/litter) / (number of viable fetuses/litter)) x 100%

Historical control data:

Historical control data from the same testing laboratories in 2014-2015 are available (see under "Any other information on results incl. tables').

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Rales and piloerection were noted at 100 mg/kg bw/day for five and one female(s), respectively. Although these signs recovered after 1-5 days, they might be related to treatment with test item.
Salivation was noted for all females at 100 mg/kg bw/day, compared to none in the other groups. This finding was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).
Moreover, alopecia was noted for one single female at 15 mg/kg bwd/day, which was not considered to be treatment related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Treatment at 100 mg/kg bw/day resulted in two unscheduled deaths.
One female at 100 mg/kg bw/day was sacrificed in extremis on Day 7 post-coitum (before dosing), the day after having received the first dose. Observations preceding early sacrifice included squeaking, rales, labored (severe) and shallow (moderate) respiration, gasping and hypothermia. At necropsy, dark red foci were noted on the thymus. This female was pregnant and had 8 normal implantations in development.
Another female of the 100 mg/kg bw/day group was found dead on Day 21 post-coitum, the day of planned necropsy. No toxicologically relevant clinical signs were noted for this female. Body weight and food consumption was slightly lower for this female, when compared to the other females of the 100 mg/kg bw/day group. This was considered to be (partly) caused by the number of fetuses, as she had only 3 (dead) fetuses and 4 early resorptions. No macroscopic alterations were noted at necropsy.
Although these mortality findings were incidental, it could not be excluded that these unscheduled deaths were related to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a trend towards lower body weights at 100 mg/kg bw/day from Day 12 post-coitum onwards. Body weight gain was statistically significantly lower at 100 mg/kg bw/day than controls on Days 15 and 18 post-coitum. For uterus corrected body weight gain was considered to be unaffected by treatment up to 100 mg/kg bw/day.
Mean body weight and body weight gain at 15 and 40 mg/kg bw/day remained in the same range as controls.
One female from the control group and three females from the 100 mg/kg bw/day group had significantly lower body weights and weight gains when compared to the other females of the same group. This was considered to be (partly) due to the relatively low number of fetuses, as two females had 1 fetus, and two other females had 3 fetuses. In addition, for two females (one with one fetus and one with two fetuses) no body weight gain was observed over Days 12 to 21 post-coitum, which was considered to be a direct treatment-related effect rather than only caused by the low number of fetuses.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption before and after correction for body weight was statistically significantly reduced at 100 mg/kg bw/day on Days 9 to 12 post-coitum. This recovered during the remainder of the treatment period.
At 15 and 40 mg/kg bw/day, food consumption before or after correction for body weight were similar as controls.

Food efficiency:

not examined

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

no effects observed

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):

Alopecia was noted for one single female at 15 mg/kg bw/day, confirming the clinical sign observed during the in-life phase.
Dark red foci on the thymus were noted for one female at 100 mg/kg bw/day, which was early sacrificed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Pre- or post-implantation loss were unaffected by treatment up to and including 100 mg/kg bw/day.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

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:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significantly lower numbers of corpora lutea (10.7 per female) were noted in the 100 mg/kg bw/day group when compared to the concurrent control group (12.2 per female). As treatment started from implantation onwards, i.e. Day 6 post-coitum, this was not considered to be treatment related. Consequently, the number of implantation sites at 100 mg/kg bw/day were slightly lower than controls as well (9.6 versus 11.4 per female). This was not statistically significant and within the range of available historical control data and not considered to be treatment related.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

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 fetal body weights (both sexes) noted by treatment up to 100 mg/kg bw/day.
Mean combined (male and female) fetal body weights were 5.3, 5.3, 5.2 and 5.4 gram for the control, 15, 40 and 100 mg/kg groups, respectively
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

Mean litter sizes were 10.7, 10.7, 11.0 and 9.1 viable fetuses/litter for the control, 15, 40 and 100 mg/kg bw/day groups, respectively.
The slightly lower number of fetuses/litter at 100 mg/kg bw/day when compared to controls was not statistically significant and within the range of available historical control data. As it was related to the lower numbers of corpora lutea and implantation sites at 100 mg/kg bw/day, it was not considered to be treatment related.
There were two females, one in the control group and one in the 100 mg/kg bw/day group, which only had one viable fetus. This was considered to be chance findings.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to 100 mg/kg bw/day.
Mean sex ratios (males:females) were 55:45, 49:51, 43:57 and 50:50 for the control, 15, 40 and 100 mg/kg groups, respectively.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on litter size of any group.
Mean litter sizes were 10.7, 10.7, 11.0 and 9.1 viable fetuses/litter for the control, 15, 40 and 100 mg/kg groups, respectively.
The slightly lower number of fetuses/litter at 100 mg/kg bw/day when compared to controls was not statistically significant and within the range of available historical control data. As it was related to the lower numbers of corpora lutea and implantation sites at 100 mg/kg bw/day, it was not considered to be treatment related.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on external morphology following treatment up to 100 mg/kg bw/day.
External malformations occurred in all groups. A small lower jaw was observed in one fetus of 100 mg/kg bw/day group and was skeletally confirmed. An absent tail combined with anal atresia were found in one fetus of 40 mg/kg bw/day group, and in 15 mg/kg bw/day group an omphalocele in one fetus and anasarca in another fetus were noticed. Omphalocele and anasarca were observed in single control fetuses as well. The single occurrence and/or group distribution of the above malformations did not indicate a treatment relationship and therefore all were considered to be chance findings.
External variations were not seen in any group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on skeletal morphology following treatment up to 100 mg/kg bw/day.
Malformations were observed in two fetuses of 100 mg/kg bw/day group and single fetuses of control, 15 and 40 mg/kg bw/day groups. A fetus from the 100 mg/kg bw/day group (which had a small lower jaw and a variety of visceral malformations) appeared to have severely malaligned sternebrae and a bent scapula as well. Another fetus of the 100 mg/kg bw/day group had malpositioned metatarsals.
The other affected fetuses (control, 15 and 40 mg/kg bw/day groups, respectively) all had a vertebral anomaly with or without associated rib anomaly. In addition, the fetus from the 15 mg/kg bw/day group (that also had an omphalocele and malpositioned kidneys) had a sternal anomaly and bent limb bones as well. The group distribution and/or single occurrence of these malformations did not suggest any treatment relationship and therefore all were considered to be chance findings.
Skeletal variations occurred at an incidence of 86.7%, 89.3%, 78.5% and 76.0% per litter in controls, 15, 40 and 100 mg/kg bw/day, respectively. All the ones noted, were not considered treatment-related, as they occurred in the absence of a dose-dependent relationship, infrequently and/or at frequencies that were within the range of available historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on visceral morphology following treatment up to 100 mg/kg bw/day.
In total five fetuses were viscerally malformed in this study. The two fetuses of 15 mg/kg bw/day group, one fetus of the 100 mg/kg bw/day group and one control fetus that were affected externally, appeared to have one or more visceral malformations as well. The other affected fetus was a 40 mg/kg bw/day group fetus. Due to the group distribution and variety of malformation observed, all were considered not to be toxicologically relevant.
Visceral variations were observed in 10.3%, 9.1%, 7.3% and 7.5% of fetuses per litter in controls, 15, 40 and 100 mg/kg bw/day groups, respectively. These all occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within the range of available historical control data.

Other effects:

no effects observed

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment-related effects observed at the highest tested dose.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Analytical verification of the tested formulations

The concentrations analyzed in the formulations were in agreement with the target concentrations (mean accuracies 100.9% (n = 6), 98.4 (n = 2) and 99.3 (n = 6) for 15, 40 and 100 mg/kg bw/day dose solutions).

The formulations of low- and high-dose groups were homogeneous ( coefficient of variation 0.6 and 1.0, respectively (n = 6)).

Summary of developmental effects observed in the study

Dose level (mg/kg bw/day)	0	8	25	75
Pregnant/total dams	22/22	22/22	22/22	22/22
-early resorptions -late resorptions (% per litter)	7.1 0.4	5.5 0.0	6.2 0.3	4.6 0.0
Dams with abortion, early deliveries, stillbirths, resorptions only and/or dead fetuses only	0	0	0	0
Pre-implantation loss (number and percent)	19 (6.2%)	26 (9.3%)	26 (9.1%)	21 (10.7%)
Post-implantation loss (number and percent)	14 (7.5%)	14 (5.5%)	19 (6.5%)	10 (4.6%)
Body weight on day 21	327	326	326	313
Body weight gain day 6-21 (%)	48	47	47	43
Gravid uterine weight (g)	76.5	76.2	76.4	65.3
Mean live offspring (number)	10.7	10.7	11.0	9.1
Live offspring (percent)	92.5	94.5	93.5	95.4
Mean fetal/pup body weight males (g)	5.5	5.5	5.3	5.5
Mean fetal body weight females	5.1	5.2	5.1	5.2
Mean fetal body weight (sexes combined)	5.3	5.3	5.2	5.4
Malformations (including runts) number and percent of fetuses per litter	3 (1.4%)	2 (0.9%)	3 (2.3%)	2 (1.5%)
Variations (% per litter) -external -soft tissue -skeletal	0 10.3 86.7	0 9.1 89.3	0 7.3 78.5	0 7.5 76.0

Historical control data (2014-2015)

Historical Control Data Rat: Crl:WI(Han) (outbred, SPF-Quality)
Gestation Day 21		Mean of Study Means
Study Date Range: 2014 - 2015
Endpoint	Total	Mean	SD	Median	Min	Max	P5	P95
No of Studies	13
Total No. of Animals in the Control Group	304
No. of Animals that Died	0
No. of Animals that were Euthanized	0
No. of Animals that Aborted or Delivered	3
Percent Pregnant		98.8	2.73	100.0	90.9	100.0	97.1	100.0
No. of Animals Examined at Laparohysterectomy	301
No. Nongravid	4
No. Gravid	297
No. with Only Resorptions	2
No. of Dams with Live Fetuses	295
Mean No. Viable Fetuses/Dam		10.7	0.71	10.6	9.1	11.6	10.3	11.2
Total No. Viable Fetuses	3194
Viable Fetuses (%/Litter)		95.2	2.63	95.9	88.9	98.4	93.6	96.8
Mean No. Postimplantation Loss/Dam		0.5	0.15	0.4	0.2	0.7	0.4	0.6
Total No. Postimplantation Losses	134
Postimplantation Loss (%/Litter)		4.8	2.63	4.1	1.6	11.1	3.2	6.4
Dead Fetuses (%/Litter)		0.0	0.11	0.0	0.0	0.4	0.0	0.1
Early Resorptions (%/Litter)		4.7	2.62	4.1	1.6	11.1	3.2	6.3
Late Resorptions (%/Litter)		0.0	0.11	0.0	0.0	0.4	0.0	0.1
Mean No. Implantations/Dam		11.2	0.69	11.1	9.6	12.0	10.8	11.6
Mean No. Corpora Lutea/Dam		11.9	0.71	11.7	10.9	13.2	11.5	12.3
Mean No. Preimplantation Loss/Dam		0.7	0.32	0.6	0.2	1.3	0.5	0.9
Total No. Preimplantation Losses	207
Preimplantation Loss (%/Litter)		6.2	3.43	5.8	2.0	14.5	4.2	8.3
Total No. Male Fetuses	1617
Total No. Female Fetuses	1577
% Males/Litter		50.8	2.12	50.7	46.6	53.7	49.5	52.0
% Female/Litter		49.2	2.12	49.3	46.3	53.4	48.0	50.5
Mean Fetal Body Weight (g)		5.2	0.08	5.2	5.1	5.3	5.1	5.2
Mean Male Body Weight (g)		5.4	0.10	5.4	5.2	5.5	5.3	5.4
Mean Female Body Weight (g)		5.1	0.07	5.1	5.0	5.2	5.0	5.1
Mean Male Placenta Weight (g)^1		0.46	0.01	0.47	0.44	0.47	0.4	0.5
Mean Female Placenta Weight (g)^1		0.44	0.01	0.44	0.42	0.45	0.4	0.5

 

¹Based on 4 datasets

Historical Control Data Rat: Crl:WI(Han) (outbred, SPF-Quality)
Gestation Day 21
Study Date Range: 2014 - 2015
No. of Studies	13
Total No. Fetuses/Litters Examined Externally	3194	295
Total No. Fetuses/Litters Examined Viscerally	2061	295
Total No. Fetuses/Litters Examined Skeletally	2059	295
	Mean of Study Means							Summary Incidence
	(% Per Litter Basis)							(Total No. Affected)
MALFORMATIONS	Mean	SD	Median	Min	Max	P5	P95	Fetuses	Litters
Total External Malformations								1	1
Total Visceral Malformations								7	7
Total Skeletal Malformations								15	15
Total Malformations								22	22
EXTERNAL
Exencephaly	0.0	0.14	0.0	0.0	0.5	0.0	0.1	1	1
Eye(s)- Open	0.0	0.14	0.0	0.0	0.5	0.0	0.1	1	1
VISCERAL
Diaphragmatic Hernia	0.0	0.08	0.0	0.0	0.3	0.0	0.1	1	1
Eye(s)- Absent and/or Small	0.1	0.26	0.0	0.0	0.9	0.0	0.2	3	3
Hydrocephaly- External	0.0	0.12	0.0	0.0	0.5	0.0	0.1	1	1
Situs Inversus	0.2	0.34	0.0	0.0	1.0	0.0	0.4	3	3
SKELETAL
Jaw- Upper Jaw Small	0.1	0.22	0.0	0.0	0.8	0.0	0.2	1	1
Jaw- Lower Jaw Absent or Small	0.1	0.22	0.0	0.0	0.8	0.0	0.2	1	1
Limb Bone(s)- Bent	0.3	0.44	0.0	0.0	1.1	0.0	0.5	4	4
Rib Anomaly	0.1	0.31	0.0	0.0	1.1	0.0	0.3	1	1
Skull Anomaly	0.1	0.34	0.0	0.0	1.2	0.0	0.3	2	2
Sternebra(e)- Fused	0.1	0.29	0.0	0.0	1.0	0.0	0.3	2	2
Sternebra(e) Malaligned (Severe)	0.0	0.08	0.0	0.0	0.3	0.0	0.1	1	1
Sternoschisis	0.1	0.22	0.0	0.0	0.8	0.0	0.2	1	1
Vertebral Anomaly With or Without Associated Rib Anomaly	0.2	0.53	0.0	0.0	1.9	0.0	0.5	3	3
Vertebral Centra Anomaly	0.1	0.22	0.0	0.0	0.8	0.0	0.2	1	1

Historical Control Data Rat: Crl:WI(Han) (outbred. SPF-Quality)
Gestation Day 21
	Mean of Study Means							Summary Incidence
	(% Per Litter Basis)							(Total No. Affected)
VARIATIONS	Mean	SD	Median	Min	Max	P5	P95	Fetuses	Litters
EXTERNAL
None Observed
VISCERAL
Kidney(s)- Renal Papilla(e) Absent and/or Small	0.1	0.25	0.0	0.0	0.9	0.0	0.2	2	2
Liver- Appendix	1.2	0.56	1.3	0.3	2.3	0.9	1.6	23	21
Liver- Discolored	0.1	0.30	0.0	0.0	1.0	0.0	0.3	3	3
Liver- Small Supernumerary Lobe(s)	4.0	1.96	4.0	1.3	7.7	2.8	5.2	69	58
Spleen- Supernumerary	0.0	0.14	0.0	0.0	0.5	0.0	0.1	1	1
Thymus- Partially Undescended Horn(s)	1.3	1.55	0.8	0.0	4.3	0.3	2.2	34	23
Thyroid- Discolored	0.1	0.36	0.0	0.0	1.3	0.0	0.3	1	1
Ureter(s)- Convoluted	1.0	2.39	0.0	0.0	8.7	0.0	2.5	43	28
Ureter(s)- Dilated	0.9	2.33	0.0	0.0	8.5	0.0	2.3	44	19
SKELETAL
7th Cervical Rudimentary Rib(s)	1.7	1.34	1.2	0.0	4.4	0.9	2.5	30	26
7th Cervical Full Rib(s)	0.1	0.36	0.0	0.0	1.1	0.0	0.4	2	2
14th Full Rib(s)	5.7	4.65	5.2	0.0	13.1	2.9	8.5	88	64
14th Rudimentary Rib(s)	44.1	19.84	54.4	19.0	72.0	32.1	56.1	798	250
Metacarpal(s) and/or Metatarsal(s) Unossified	2.2	1.97	1.0	0.0	6.3	1.0	3.4	41	24
Pelvic Girdle- Caudal Shift	6.6	3.77	7.1	1.7	12.8	4.3	8.9	127	71
Rib(s)- Bent	10.6	7.78	10.2	0.8	22.3	5.9	15.3	162	85
Rib(s)- Short	0.0	0.06	0.0	0.0	0.2	0.0	0.0	1	1
Skull- Reduced Ossification	2.7	2.55	1.8	0.0	7.0	1.2	4.3	81	46
Skull- Supernumerary Site	0.0	0.14	0.0	0.0	0.5	0.0	0.1	1	1
Sternebra(e) #1, #2, #3 and/or #4 Unossified	0.2	0.31	0.0	0.0	0.8	0.0	0.3	3	3
Sternebra(e) #5 and/or #6 Unossified	0.9	1.33	0.0	0.0	4.1	0.1	1.7	37	23
Sternebrae- Malaligned (Slight or Moderate)	11.1	5.72	8.9	4.4	21.3	7.6	14.5	188	131
Sternum- Supernumerary Ossification Site	0.1	0.31	0.0	0.0	1.1	0.0	0.3	1	1
Vertebral Centra- Reduced Ossification	0.6	0.88	0.4	0.0	3.0	0.1	1.2	12	12

Applicant's summary and conclusion

Conclusions:

In a GLP-compliant guideline study with rats, the administration of the test substance by oral gavage to pregnant rats during gestation days 6-21 did not result in developmental effects on the offspring at the highest tested dose of 100 mg/kg bw/day. This level was considered to be a NOAEL for developmental effects. Based on the clinical signs and reduced body weight gain at the highest dose level in maternal animals, the NOAEL for maternal toxicity was set at 40 mg/kg bw/day.

Executive summary:

In a GLP-compliant OECD guideline 414 study, the test substance was administered to groups of 22 pregnant rats at dose levels of 0 (concurrent vehicle controls), 15, 40 and 100 mg/kg bw/day. Two mortalities occurred at the highest dose level (day 7 and day 21); however, in the absence of corroborative gross pathological and histopathological findings these deaths are likely to have been incidental. Rales and piloerection were observed in 5 and 1 female of the high-dose group, respectively, but disappeared after 1 -5 days. Lower body weight gains were observed on days 15 and 18 of gestation. Based on these effects the maternal NOAEL was set at 40 mg/kg bw/day.

Statistically significantly lower numbers of corpora lutea were noted in the high-dose group in comparison to the controls, resulting in a lower number of implantation sites; however, as the treatment started after the implantation, i.e. from day 6 post-coitum, this finding was considered to be not related to the treatment. Furthermore, the number of implantation sites was within the range of historical control data. There were no effects on the number of early and late resorption, post-implantation losses or the number of viable fetuses. Body weights and sex ratio of fetuses were unaffected by the treatment. There were no treatment-related external, visceral or skeletal variations or

malformations at any dose level. Based on this, the NOAEL for developmental toxicity was set at the highest tested dose of 100 mg/kg bw/day.