Hexanoic acid, 6-[(3,5,5-trimethyl-1-oxohexyl)amino]-, compd. with 2,2',2''-nitrilotris[ethanol] (1:1)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Remarks:

Prenatal Developmental Toxicity as per OECD414, adopted on 25 June 2018

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 September 2018 to 07 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD414, adopted on 25 June 2018

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted on 25 June 2018

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

tris(2-hydroxyethyl)ammonium 6-(3,5,5-trimethylhexanamido)hexanoate

EC Number:

701-138-0

Cas Number:

242482-67-3

Molecular formula:

C21H46N2O6

IUPAC Name:

tris(2-hydroxyethyl)ammonium 6-(3,5,5-trimethylhexanamido)hexanoate

Test material form:

liquid: viscous

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Clariant Produkte and 18-UH-0703
- Expiration date of the batch: March 2020
- Purity test date: 24 March 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Stability under test conditions: The test item formulations at 1 and 100 mg/mL were stable up to 6 hours at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Clearly miscible with water at the concentration of 50 mg/mL stable upto 6 hours in ambient condition.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Miscible with water to get desired concentration of 5,15 and 50 mg/mL.


FORM AS APPLIED IN THE TEST (if different from that of starting material) :Liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred
- Age at study initiation: 10 weeks
- Weight at study initiation: 200 to 300 grams
- Housing: Pre-mating: Maximum of three animals of same sex; Mating: 1:1 ratio; Post-mating: Males: Maximum of three animals of same sex and Females: Individual housing
- Diet (ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG)
- Water (ad libitum): Deep bore-well water passed through reverse osmosis unit
- Acclimation period: Minimum of five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 to 22.9
- Humidity (%): 48% to 64%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 03 September 2018; To: 06 November 2018

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Not applicable

Remarks on MMAD:

Not applicable

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:


VEHICLE
- Justification for use and choice of vehicle: The test item was clearly miscible with distilled water at the concentration of 50 mg/mL (highest dose concentration). Hence, distilled water was selected as a vehicle for test item formulation preparations. Distilled water is acceptable and routinely used for toxicity studies.

- Concentration in vehicle: Low dose: 5 mg/mL; Mid dose: 15 mg/mL; High dose: 50 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight
- Purity: 100%

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation analysis for dose concentration verification was done by Analytical Chemistry department of Bioneeds India Private Limited. Sampling and analysis of formulations were performed during first week and last week of the treatment. The samples were collected in duplicates from vehicle control, low, mid and high dose concentrations.
The collected samples were transferred to Analytical Chemistry department of Bioneeds India Private Limited for dose concentration analysis. One set of aliquot of each formulation was analyzed. The second aliquot was stored as a backup purpose at established stability conditions. The second set of samples was discarded, as the analysis results of first set of samples were within the limits. Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10%.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 days
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Gestation day 5 to 19 [15 days for each animal]

Frequency of treatment:

Once daily

Duration of test:

4 months

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day

Remarks:

Mid dose

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day

Remarks:

High dose

No. of animals per sex per dose:

25 mated presumed-pregnant females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses of 0, 50, 150 and 500 mg/kg body weight for vehicle control, low dose, mid dose and high dose respectively were selected in consultation with the sponsor based on the results of dose range finding study for Prenatal developmental Toxicity study of 6-(isononanoylamino)hexanoic acid, compound with 2,2ꞌ,2ꞌꞌ- nitrilotriethanol (1:1) by oral gavage in Sprague Dawley Rats (Bioneeds Study No.: BIO-TX 3500).
These doses were selected as the test item when administered orally to mated and presumed-pregnant Sprague Dawley females from Gestation Day [GD] 5 to 19 and sacrificed on Gestation Day [GD] 20. The NOAEL for maternal toxicity was 150 mg/kg due to occurrence of reduced litter size and increased early resorptions at 500 mg/kg. The NOAEL for the developmental toxicity was 500 mg/kg, the high dose, due to no evidence of adverse effects on fetal developmental or incidences of external and soft tissue anomalies.

- Rationale for animal assignment (if not random): The body weight of mated females on each day of gestation day ‘0’ was recorded and arranged in the ascending order of their body weight until required number of mated females acquired for each group. These mated females were evenly distributed to all the groups based on their body weights so as to maintain comparable mean body weight for all groups and permanent identification numbers were assigned.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
- Cage side observations checked in table [1] were included.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation Day 0, 3, 5, 8, 11, 14, 17, 19 and on 20 (day of caesarean section).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: Yes, Gestation Day 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17, 17 to 19 and 19 to 20 (day of caesarean section).


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: All sysytemic organs and tissue along with reproductiver organs

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
- Other: The weight of thyroid along with the parathyroid was recorded post-fixation

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:

- Data was subjected to various statistical analysis using SPSS software version 22. All analyses and comparisons were evaluated at the 95% level of confidence (P<0.05),
- Data of non-pregnant females was not included in mean calculation and statistical analysis.

One-way ANOVA with Dunnett’s post test - Gestation body weight (g), Percent change in gestation body weight, Corrected body weight (g & %), Gravid uterus weight (g), Feed consumption (g), Fetal weights (g), Anogenital distance of all live fetuses (mm), Crown-rump length of fetuses (mm), serum T4, T3 and TSH lelvels.

Kruskal-Wallis (Non-parametric) - No. of corpora lutea, implantations, resorptions, litter size, sex ratio, implantation lossess, live/dead fetuses.

Cross Tabs Chi-square test/ Fischer's Exact Test - Pregnancy rate, No. of dams with/without live/dead fetuses, No. of dams with/without lresorptions

Indices:

Corrected Body weight (g) = (Gestation day 20 body weight - Gestation day 5 body weight) - Gravid uterus weight.

Percent of Live/Dead Fetuses = (Number of Live/Dead Fetuses / Litter Size) x 100.

Percent of Early/Late Resorptions (%) = (Number of Early/late Resorptions/ Number of Implantations) x 100.

Early Resorptions per Group (%) = (Number of Dams with Early Resorptions in the group / Total Number of Dams in the Group) x 100.

Pre-implantation Loss (%) = (Number of Corpora lutea - Number of implants) / Number of Corpora lutea x 100.

Post-implantation Loss (%) per Dam = (Number of Dams with Post-implantation loss / Total Number of Dams) x 100

Male/Female Sex Ratio = Number of live male fetuses / Number of live female fetuses.

Male/Female Fetuses (%) = (Number of live male/female fetuses / Total number of live fetuses) x 100.

Group/Litter Fetal Observation for External, Visceral and Skeletal Examinations:

Fetal incidence (%) = (Number of Fetuses with a particular observation / Total number of Fetuses in a group) x 100

Historical control data:

Not applicable

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity observed at any of the tested dose group animals (50, 150 and 500 mg/kg body weight) during the experimental period.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable

Mortality:

no mortality observed

Description (incidence):

There were no mortality/morbidity observed at any of the tested dose group animals (50, 150 and 500 mg/kg body weight) during the experimental period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes noted in body weight and percent change in body weight during gestation period across all the tested dose groups (50, 150 and 500 mg/kg body weight) when compared with the vehicle control group.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes noted in average feed consumption during gestation period across all the tested dose groups (50, 150 and 500 mg/kg body weight) when compared with the vehicle control group. The average feed consumption (g/animal/day) across groups was comparable.
A statistically significant reduction in average feed consumption during gestation day 11 to 14 in G4 group animals was observed. This sporadic incidence is considered as incidental and unrelated to treatment with test item due to unaffected body weights during this period.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable

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

not examined

Description (incidence and severity):

Not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

Not applicable

Haematological findings:

not examined

Description (incidence and severity):

Not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable

Immunological findings:

not examined

Description (incidence and severity):

Not applicable

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The absolute thyroid along with parathyroid weights (g) were 0.0246, 0.0245, 0.0238 and 0.0249 and relative thyroid along with parathyroid weights (%) were 0.0062, 0.0062, 0.0061 and 0.0062 for G1 (0 mg/kg), G2 (50 mg/kg), G3 (150 mg/kg) and G4 (500 mg/kg) respectively. There were no statistically significant differences in mean absolute and relative thyroid along with parathyroid weight and no treatment related changes for this parameter across the dose groups when compared to controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes noted in any of the dose group (50, 150 and 500 mg/kg body weight) and vehicle control group animals (0 mg/kg body weight) during conduct of the necropsy.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The histopathology of thyroid along with parathyroid was conducted for all the dose group dams (50, 150 and 500 mg/kg body weight) and vehicle control group animals (0 mg/kg body weight). There were no treatment related histopathological findings noticed in the in any of the dose group animals during conduct of the histopathological examination in the study.
Presence of ultimobranchial cysts in G1 (2 incidences), G2 (1 incidence), G3 (4 incidences) and G4 (3 incidences); ectopic thymus in G1 (1 incidence) and G4 (3 incidences) were congenital lesions and it does not have toxicological significance.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Not applicable

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Serum Triiodothyronine (T3) Levels: The serum T3 levels (ng/mL) were 2.823, 2.813, 2.749 and 2.469 for G1 (0 mg/kg), G2 (50 mg/kg), G3 (150 mg/kg) and G4 (500 mg/kg) respectively. There were no changes in serum T3 levels (ng/mL) across all the tested dose groups when compared with the vehicle control group. The values obtained across groups were comparable. A statistically significant reduction in serum T3 levels (ng/mL) in G4 group animals was observed. This incidence is considered as incidental and unrelated to treatment with test item due to comparable values across groups and the values obtained were within biological control range.

Serum Thyroxine (T4) Levels: The serum T4 levels (ng/mL) were 73.380, 71.373, 67.687 and 64.964 for G1 (0 mg/kg), G2 (50 mg/kg), G3 (150 mg/kg) and G4 (500 mg/kg) respectively. There were no changes in serum T4 levels (ng/mL) across all the tested dose groups when compared with the vehicle control group. The values obtained across groups were comparable. A statistically significant reduction in serum T4 levels (ng/mL) in G3 and G4 group animals was observed. This incidence is considered as incidental and unrelated to treatment with test item due to comparable values across groups and the values obtained were within biological control range.


Serum Thyroid Stimulating Hormone (TSH) Levels: The serum TSH levels (µIU/mL) were 5.717, 5.539, 5.171 and 4.873 for G1 (0 mg/kg), G2 (50 mg/kg), G3 (150 mg/kg) and G4 (500 mg/kg) respectively. There were no statistically significant differences in serum TSH levels and no treatment related changes for this parameter across the dose groups when compared to controls.

Details on results:

The Test item, 6-(Isononanoylamino) hexanoic acid, compound with 2, 2′, 2″-nitrilotriethanol (1:1) was administered via oral route at a dose volume of 10 mL/kg body weight to mated female rats from gestation day 5 (GD5) through gestation day 19 (GD19). The dose levels were 0 mg/kg, 50 mg/kg, 150 mg/kg and 500 mg/kg for the control (G1), low (G2), mid (G3) and high (G4) groups, respectively.

The results of the experiment support the conclusion that the NOAEL [No Observed Adverse Effect Level] for the test item, 6-(Isononanoylamino) hexanoic acid, compound with 2, 2′, 2″-nitrilotriethanol (1:1) for maternal and developmental toxicity toxicity is 500 mg/kg, the high dose, as there were no effects on maternal parameters as well as fetal parameters at all the tested dose groups.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions were noted

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Pre-Implantation Loss: The mean percentage pre-implantation loss was 0.00, 0.00, 0.00 and 1.92 for G1 (0 mg/kg), G2 (50 mg/kg), G3 (150 mg/kg) and G4 (500 mg/kg) respectively. There were no statistically significant differences in these values and no treatment related changes for this parameter across the dose groups when compared to controls.

Post-Implantation Loss: The mean percentage of post-implantation loss was 6.89, 4.57, 9.10 and 7.75 for G1 (0 mg/kg), G2 (50 mg/kg), G3 (150 mg/kg) and G4 (500 mg/kg) respectively. There were no statistically significant differences in these values and no treatment related changes for this parameter across the dose groups when compared to controls.

Overall Implantation Loss Assessment: The occurrence of implantation losses across all groups is within the historical control range for studies with this species and strain. There is no dose-related trend in the values so these losses were considered as incidental and unrelated to treatment. Dosing of test item, 6-(Isononanoylamino) hexanoic acid, compound with 2,2′,2″-nitrilotriethanol (1:1) to pregnant rats began on GD5. Implantation occurs in rats on or about GD5. For this reason any pre-implantation loss which occurred prior to the initiation of dosing cannot be treatment related.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No such incidences noted at all the dose group dams (50, 150 and 500 mg/kg body weight) and vehicle control group animals (0 mg/kg body weight)

Early or late resorptions:

no effects observed

Description (incidence and severity):

Early Resorptions: The mean number of early resorptions per dam was 0.78, 0.54, 0.86 and 0.17 and percentage of early resorptions was 5.37, 4.31, 7.79 and 4.81 for G1 (0 mg/kg), G2 (50 mg/kg), G3 (150 mg/kg) and G4 (500 mg/kg) respectively. There were no statistically significant differences observed in the number and percentage of early resorptions per dam across dose groups when compared to the vehicle control.

Late Resorptions: The mean number of late resorptions per dam was 0.22, 0.04, 0.17 and 0.33 and percentage of late resorptions was 1.52, 0.26, 1.30 and 2.94 for G1 (0 mg/kg), G2 (50 mg/kg), G3 (150 mg/kg) and G4 (500 mg/kg) respectively. There were no statistically significant differences observed in the number and percentage of late resorptions per dam across dose groups when compared to the vehicle control.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no dead fetuses noted for any litter in any of the test dose group (50, 150 and 500 mg/kg body weight) or the vehicle control litters (0 mg/kg body weight).

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

All dams were sacrificed on gestation day 20
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): All dams were sacrificed on gestation day 20

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

G1 (0 mg/kg body weight): Pregnancy rate = 92.0%;
G2 (50 mg/kg body weight): Pregnancy rate = 96.0%;
G3 (150 mg/kg body weight): Pregnancy rate = 92.0%;
G4 (500 mg/kg body weight): Pregnancy rate = 96.0%.

Other effects:

no effects observed

Description (incidence and severity):

Gravid Uterus Weight and Corrected Body Weight: There were no changes noted in gravid uterus weight and body weights corrected for maternal weight across all the tested dose groups (50, 150 and 500 mg/kg body weight) when compared with the vehicle control group.

Details on maternal toxic effects:

The Test item, 6-(Isononanoylamino) hexanoic acid, compound with 2, 2′, 2″-nitrilotriethanol (1:1) was administered via oral route at a dose volume of 10 mL/kg body weight to mated female rats from gestation day 5 (GD5) through gestation day 19 (GD19). The dose levels were 0 mg/kg, 50 mg/kg, 150 mg/kg and 500 mg/kg for the control (G1), low (G2), mid (G3) and high (G4) groups, respectively.
The results of the experiment support the conclusion that the NOAEL [No Observed Adverse Effect Level] for the test item, 6-(Isononanoylamino) hexanoic acid, compound with 2, 2′, 2″-nitrilotriethanol (1:1) for maternal toxicity is 500 mg/kg, the high dose, as there were no effects on maternal parameters as well as fetal parameters at all the tested dose groups.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

maternal abnormalities

mortality

necropsy findings

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

other: Thyroid hormonal assay results [T3, T4 and TSH]

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Localisation:

amniotic fluid

cervix

mammary gland

ovary

oviduct

placenta

umbilical cord

uterus

vagina

vitreous chamber / humor

other: thyroid

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

For groups G1 (0 mg/kg), G2 (50 mg/kg), G3 (150 mg/kg) and G4 (500 mg/kg), respectively, the male mean fetal weights were 4.35g, 4.19g, 4.07g and 4.17g; the female mean fetal weights were 3.99g, 3.99g, 3.80g and 3.89g. There were no changes in mean fetal weights across all the tested dose groups when compared with the vehicle control group. The values obtained across groups were comparable. A statistically significant reduction in mean male and female fetal weights in G3 group was observed. This incidence is considered as incidental and unrelated to treatment with test item due to comparable values across groups and also other fetal parameters are unaffected.

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): For groups G1 (0 mg/kg), G2 (50 mg/kg), G3 (150 mg/kg) and G4 (500 mg/kg), respectively, the male mean fetal weights were 4.35g, 4.19g, 4.07g and 4.17g; the female mean fetal weights were 3.99g, 3.99g, 3.80g and 3.89g. There were no changes in mean fetal weights across all the tested dose groups when compared with the vehicle control group. The values obtained across groups were comparable. A statistically significant reduction in mean male and female fetal weights in G3 group was observed. This incidence is considered as incidental and unrelated to treatment with test item due to comparable values across groups and also other fetal parameters are unaffected.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The mean litter sizes, assessed as in the utero total of fetuses [live plus dead] per dam, were 12.17, 12.00, 11.48 and 11.54 for groups G1 (0 mg/kg), G2 (50 mg/kg), G3 (150 mg/kg) and G4 (500 mg/kg) respectively. There were no dead fetuses within any litter in any group, therefore, the numbers of live fetuses per dam across groups were the same. There were no statistically significant differences noted across dose groups when compared to the control.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex ratio calculated as the mean percentage of male fetuses was 1.61, 1.41, 1.43 and 1.22 for G1 (0 mg/kg), G2 (50 mg/kg), G3 (150 mg/kg) and G4 (500 mg/kg) respectively. There were no statistically significant differences in these values and no treatment related changes for this parameter across the dose groups when compared to controls.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean litter sizes, assessed as in the utero total of fetuses [live plus dead] per dam, were 12.17, 12.00, 11.48 and 11.54 for groups G1 (0 mg/kg), G2 (50 mg/kg), G3 (150 mg/kg) and G4 (500 mg/kg) respectively. There were no dead fetuses within any litter in any group, therefore, the numbers of live fetuses per dam across groups were the same. There were no statistically significant differences noted across dose groups when compared to the control.

Changes in postnatal survival:

not specified

Description (incidence and severity):

Not applicable, all fetuses were sacrificed on GD 20

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

A total of 280 (23), 288 (24), 238 (23) and 277 (24) fetuses (litters) with a mean of 12.17, 12.00, 11.48 and 11.54 fetuses per dam were available for gross external examination from groups G1 (0 mg/kg), G2 (50 mg/kg), G3 (150 mg/kg) and G4 (500 mg/kg), respectively. No treatment related external abnormalities were noted within any group during external examination of these fetuses. However, the following variations were noted from all the group fetuses during fetal external examinations:

In G1 (vehicle control) group, haemorrhagic spot on the right fore limb in one fetus, haemorrhagic spot on tail in one fetus and haemorrhagic spot on the left hind limb in one fetus were noted.
In G2 (50 mg/kg) group, haemorrhagic spot on the fore limb bilaterally in one fetus, haemorrhagic spot on right forelimb and neck in one fetus and haemorrhagic spot on the right hind limb in one fetus were noted.
In G3 (150 mg/kg) group, haemorrhagic spot on the left forelimb in one fetus, haemorrhagic spot on left hind limb in one fetus and haemorrhagic spot on the right fore limb in one fetus were noted.
In G4 (500 mg/kg) group, haemorrhagic spot on the left forelimb in one fetus, haemorrhagic spot on right forelimb in one fetus, haemorrhagic spot on the left hind limb in one fetus and haemorrhagic spot on the hind limb bilaterally in one fetus were noted.

These findings are common for fetuses of this species and strain and cannot be considered as treatment related.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were 148 (23), 150 (24), 137 (23), and 144 (24) fetuses (litters) available for skeletal examination in groups G1 (0 mg/kg), G2 (50 mg/kg), G3 (150 mg/kg) and G4 (500 mg/kg), respectively. No treatment-related malformations or variations were observed during skeletal examinations of fetuses at any dose. However, the following malformations and variations were noted from all the group fetuses during fetal skeletal examinations which were not considered as treatment related:

In G1 (vehicle control) group, the malformations like, misshapen interparietal skull bones in one fetus, absence of sternum no. 5 in three fetuses and absence of proximal phalanges no. 3 and 4 bilaterally in six fetuses were noted. The variations like, poorly ossified interparietal skull bones in one fetus, poorly ossified supra occipital skull bones in two fetuses, poorly ossified sternum no. 5 in one fetus, incompletely ossified and bipartite ossified sternum no. 5 in two fetuses each, poorly ossified sternum no. 6 in two fetuses, incompletely ossified sternum no. 5 and 6 in two fetuses, discontinuous rib no. 11 unilaterally in one fetus, wavy rib no. 13 in three fetuses, dumbbell shaped centrum no. 5 and 6 of lumbar vertebrae in one fetus, extra ossification site at arch no. 4 of sacral vertebrae in two fetuses, poorly ossified centrum no. 4 of sacral vertebrae in one fetus and extra ossification site at arch no. 2 of caudal vertebrae in one fetus were noted.

In G2 (50 mg/kg) group, the malformations like, absence of sternum no. 5 in four fetuses, absence of sternum no. 6 in one fetus and absence of proximal phanges no. 3 and 4 bilaterally in seven fetuses were noted. The variations like, poorly ossified interparietal skull bones in three fetuses, poorly ossified supra occipital skull bones in two fetuses, poorly ossified sternum no. 5 in two fetuses, poorly ossified sternum no. 6 in one fetus, incompletely ossified sternum no. 6 in six fetuses, wavy rib no. 13 in two fetuses, dumbbell shaped centrum no. 5 and 6 of lumbar vertebrae in two fetuses, dumbbell shaped centrum no. 4 of lumbar vertebrae in one fetus, poorly ossified centrum no. 4 of sacral vertebrae in one fetus and extra ossification site at arch no. 2 of caudal vertebrae in two fetuses were noted.

In G3 (150 mg/kg) group, the malformations like, absence of sternum no. 5 in six fetuses and absence of proximal phalanges no. 3 and 4 bilaterally in one fetus were noted. The variations like, poorly ossified interparietal skull bones in four fetuses, poorly ossified supra occipital skull bones in two fetuses, bipartite ossified sternum no. 5 in one fetus, incompletely ossified sternum no. 6 in six fetuses, wavy rib no. 11, 12 and 13 in one fetus, extra ossification site at arch no. 4 of sacral vertebrae in four fetuses and extra ossification site at arch no. 2 of caudal vertebrae in three fetuses were noted.

In G4 (500 mg/kg) group, absence of sternum no. 5 in two fetuses, absence of sternum no. 6 in one fetus, absence of sternum no. 5 and 6 in two fetuses and absence of proximal phalanges no. 3 and 4 bilaterally in five fetuses were note. The variations like, poorly ossified supra occipital skull bones in one fetus, incompletely ossified sternum no. 6 in two fetuses each, wavy rib no. 13 in four fetuses, wavy rib no. 11, 12 and 13 in one fetus, extra ossification site at arch no. 4 of sacral vertebrae in two fetuses and extra ossification site at arch no. 2 of caudal vertebrae in five fetuses were noted.

There were multiple skeletal malformations and variations noted. These malformations and variations are correlated in that they are anatomically related but they are not uncommon findings for fetuses of this species. In general, the incidences were of a single fetus in a litter. The malformations and variations did not occur in a dose-dependent pattern, nor was the severity or the incidence of the finding increased with dose.

The skeletal malformations reflect variations in the maturation rate of the ossified tissues of rat fetuses. They are unlikely to be detectable in juvenile animals after continued growth postnatally.

These skeletal malformations did not occur in a dose-dependent pattern, nor was the severity or the incidence of the findings increased with dose.
As with the skeletal malformations, the skeletal variations reflect variations in the maturation rate of the ossified tissues of rat fetuses and they are unlikely to be detectable in juvenile animals. The skeletal variations did not occur in a dose-dependent pattern, nor was the severity or the incidence of the findings increased with dose.

Overall, the skeletal morphologic observations reflect variations in the maturation rate of the ossified tissues of rat fetuses; they did not occur in a dose-dependent pattern, nor was the severity of the anomaly increased with dose. These result support the conclusion that the findings are incidental and that the test item did not produce an adverse effect on the skeletal system during fetal development at any dose in this study.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were 132 (23), 138 (24), 127 (23) and 133 (24) fetuses (litters) for visceral examination in groups G1 (0 mg/kg), G2 (50 mg/kg), G3 (150 mg/kg) and G4 (500 mg/kg), respectively. No treatment-related malformations or variations were observed during visceral examinations of fetuses at any dose. However, the following malformations and variations were noted from all the group fetuses during fetal visceral examinations which were not considered as treatment related:

In G1 (vehicle control) group, renal pelvis dilation unilateral (variation) in two fetuses and pale colored kidney unilaterally (variation) in one fetus were noted.

In G2 (50 mg/kg) group, cleft palate (malformation) in two fetuses and pale colored kidney unilaterally (variation) in three fetuses, dilated ventricles of brain (variation) in two fetuses and pale colored liver, kidneys and adrenals bilaterally (variation) were noted.

In G3 (150 mg/kg) group, cleft palate (malformation) in one fetus, pale colored kidneys unilaterally (variation) in three fetuses, was noted.

In G4 (500 mg/kg) group, cleft palate (malformation) in one fetus and pale colored kidney in one fetus was noted.

These observations are common findings for fetuses of this species; they did not occur in a dose-dependent pattern, nor was the severity or the incidence of the findings increased with dose. This result supports the conclusion that the findings are incidental and that the test item, 6-(Isononanoylamino) hexanoic acid, compound with 2,2′,2″-nitrilotriethanol (1:1), did not produce an adverse effect on the soft tissues during fetal development.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal Crown rump Length: For groups G1 (0 mg/kg), G2 (50 mg/kg), G3 (150 mg/kg) and G4 (500 mg/kg), respectively, the male fetal crown rump length was 39.12 mm, 38.49 mm, 37.94 mm and 38.33 mm; the female fetal crown rump length was 37.40 mm, 37.33 mm, 36.45 mm and 36.80 mm. There were no statistically significant differences in fetal crown rump length and no treatment related changes for this parameter across the dose groups when compared to controls.

Fetal Ano-genital Distance Ratio: For groups G1 (0 mg/kg), G2 (50 mg/kg), G3 (150 mg/kg) and G4 (500 mg/kg), respectively, the mean male ano-genital distance was 2.55 mm, 2.55 mm, 2.54 mm and 2.60 mm; the mean female ano-genital distance was 1.39 mm, 1.37 mm, 1.28 mm and 1.36 mm. There were no changes in mean ano-genital distance across all the tested dose groups when compared with the vehicle control group. The values obtained across groups were comparable. A statistically significant reduction in mean female ano-genital distance in G3 group was observed. This incidence is considered as incidental and unrelated to treatment with test item due to comparable values across groups and also other fetal parameters are unaffected.

Details on embryotoxic / teratogenic effects:

The Test item, 6-(Isononanoylamino) hexanoic acid, compound with 2, 2′, 2″-nitrilotriethanol (1:1) was administered via oral route at a dose volume of 10 mL/kg body weight to mated female rats from gestation day 5 (GD5) through gestation day 19 (GD19). The dose levels were 0 mg/kg, 50 mg/kg, 150 mg/kg and 500 mg/kg for the control (G1), low (G2), mid (G3) and high (G4) groups, respectively. The results of the experiment support the conclusion that the NOAEL [No Observed Adverse Effect Level] for the test item, 6-(Isononanoylamino) hexanoic acid, compound with 2, 2′, 2″-nitrilotriethanol (1:1) for fetal toxicity is 500 mg/kg, the high dose, as there were no effects on fetal parameters at all the tested dose groups.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

other: Fetal Crown rump Length and Ano-genital Distance Ratio

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Localisation:

external: cranium

external: ear

external: eye

external: face

external: limb

external: paw

external: tail

external: trunk

external: anogenital distance

external: anus

external: genital tubercle

external: large intestine

external: thorax

external: umbilicus

external: pelvic region

skeletal: skull

skeletal: skull, fontanelles

skeletal: skull sutures

skeletal: clavicle

skeletal: scapule

skeletal: forelimb

skeletal: sternum

skeletal: rib

skeletal: supernumerary rib

skeletal: vertebra

skeletal: pelvic girdle

skeletal: hindlimb

visceral/soft tissue: integumentary

visceral/soft tissue: gastrointestinal tract

visceral/soft tissue: hepatobiliary

visceral/soft tissue: urinary

visceral/soft tissue: cardiovascular

visceral/soft tissue: heamatopoietic

visceral/soft tissue: immune system

visceral/soft tissue: musculoskeletal system

visceral/soft tissue: nervous system

visceral/soft tissue: central nervous system

visceral/soft tissue: peripheral nervous system

visceral/soft tissue: somatic nervous system

visceral/soft tissue: autonomic nervous system

visceral/soft tissue: endocrine system

visceral/soft tissue: respiratory system

visceral/soft tissue: male reproductive system

visceral/soft tissue: female reproductive system

visceral/soft tissue: eye

visceral/soft tissue: ear

Overall developmental toxicity

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

500 mg/kg bw/day

Treatment related:

no

Any other information on results incl. tables

TABLE 1.        SUMMARY OF CLINICAL SIGNS OF TOXICITY AND MORTALITY RECORD

Group & Dose (mg/kg body weight)	No. of Animals	Clinical Signs of Toxicity during Gestation Period	Mortality (No. of Mortality / No. of Animals dosed)
G1 & 0	25	N	0/25
G2 & 50	25	N	0/25
G3 & 150	25	N	0/25
G4 & 500	25	N	0/25

N: Normal

TABLE 2.        SUMMARY OF GESTATION BODY WEIGHT (g)

                

Group & Dose (mg/kg body weight)		Body Weight (g) on Gestation Day
		0	3	5	8	11	14	17	19	20
G1 & 0	Mean	281.51	292.45	299.22	307.27	321.95	333.84	359.85	388.29	404.13
	±SD	28.61	31.10	32.54	32.40	32.74	32.11	32.25	36.80	37.83
	n	23	23	23	23	23	23	23	23	23
G2 & 50	Mean	275.83	288.58	293.92	302.10	317.06	331.46	355.73	381.64	396.79
	±SD	15.98	16.46	18.25	18.20	19.05	21.41	24.04	28.37	30.81
	n	24	24	24	24	24	24	24	24	24
G3 & 150	Mean	279.02	290.25	297.51	305.10	320.59	332.47	355.97	380.93	394.24
	±SD	17.56	18.45	18.64	17.80	20.19	20.45	23.81	28.39	29.76
	n	23	23	23	23	23	23	23	23	23
G4 & 500	Mean	287.16	300.56	307.45	313.68	324.94	337.97	362.02	385.91	402.84
	±SD	21.25	22.11	23.39	23.99	25.25	26.39	31.26	37.97	39.37
	n	24	24	24	24	24	24	24	24	24

SD: Standard Deviation; n: number of animals

 

TABLE 3.        SUMMARY OF PERCENT CHANGE IN GESTATION BODY WEIGHT (%) RECORD

                                                                                                            

Group & Dose (mg/kg body weight)	Percent Change in Body Weight (%) during Gestation Day
		0-3	3-5	5-8	8-11	11-14	14-17	17-19	19-20	5-20
G1 & 0	Mean	3.86	2.30	2.73	4.84	3.76	7.88	7.90	4.10	35.45
	±SD	2.35	1.48	1.41	2.07	1.76	2.90	3.50	1.52	7.79
	n	23	23	23	23	23	23	23	23	23
G2 & 50	Mean	4.65	1.83	2.80	4.97	4.53	7.33	7.26	3.96	35.06
	±SD	2.13	1.09	1.42	1.96	1.91	2.59	2.27	1.49	7.41
	n	24	24	24	24	24	24	24	24	24
G3 & 150	Mean	4.04	2.52	2.58	5.10	3.73	7.06	6.98	3.50	32.59
	±SD	2.02	1.31	1.32	3.40	1.69	2.28	2.37	1.65	7.09
	n	23	23	23	23	23	23	23	23	23
G4 & 500	Mean	4.69	2.28	2.03	3.59	4.02	7.07	6.52	4.40	30.92
	±SD	1.87	1.23	1.05	1.55	1.43	2.55	2.93	1.73	6.31
	n	24	24	24	24	24	24	24	24	24

SD: Standard Deviation; n: No. of animals

TABLE 4.        SUMMARY OF GRAVID UTERUS WEIGHT AND CORRECTED BODY WEIGHT RECORD

                                                                                                                                     

Group & Dose (mg/kg body weight)		Gravid Uterus Weight (g)	Body Weight Change (g) from GD 5 to GD 20	Corrected Body weight (g)	Corrected Body weight (%)
G1 & 0	Mean	76.30	104.91	28.62	9.79
	±SD	17.14	19.58	11.71	4.32
	n	23	23	23	23
G2 & 50	Mean	73.22	102.87	29.66	10.13
	±SD	18.45	21.96	10.63	3.73
	n	24	24	24	24
G3 & 150	Mean	67.77	96.73	28.95	9.78
	±SD	12.36	21.07	11.74	3.94
	n	23	23	23	23
G4 & 500	Mean	70.39	95.39	25.00	8.18
	±SD	22.86	21.93	7.68	2.61
	n	24	24	24	24

SD: Standard Deviation; n: No. of animals

TABLE 5.        SUMMARY OF AVERAGE FFED CONSUMPTION (g/animal/day)

                                                                                                                                                                                                                                                                                                                                                                                                                                                                Refer

Group & Dose (mg/kg body weight)		Feed Consumption on Gestation Day (g/animal/day)
		0-3	3-5	5-8	8-11	11-14	14-17	17-19	19-20
G1 & 0	Mean	17.14	22.10	19.77	21.26	23.72	25.54	27.55	26.36
	±SD	2.60	2.85	2.16	2.27	2.61	2.29	2.74	3.62
	n	23	23	23	23	23	23	23	23
G2 & 50	Mean	17.39	21.17	18.98	20.91	22.78	24.82	27.80	26.91
	±SD	2.33	2.70	2.32	1.83	2.24	2.70	3.16	3.22
	n	24	24	24	24	24	24	24	24
G3 & 150	Mean	15.96	21.93	19.03	20.65	22.45	24.69	26.23	26.79
	±SD	2.38	4.70	2.38	1.89	2.53	2.35	2.38	3.66
	n	23	23	23	23	23	23	23	23
G4 & 500	Mean	17.28	21.23	18.88	19.92	21.49*	23.94	26.31	26.42
	±SD	2.96	3.09	2.08	2.05	2.20	2.08	2.52	3.49
	n	24	24	24	24	24	24	24	24

SD: Standard Deviation; n: No. of animals

*. The mean difference is significant at the 0.05 level.

TABLE 6.        SUMMARY OF UTERI OBSERVATION RECORD

           

Group & Dose (mg/kg body weight)		No. of Corporalutea	No. of Implantations	Litter size	No. of Live Fetuses	No. of Male Live Fetuses	No. of Female Live Fetuses	No. of Dead Fetuses	No. of Early Resorptions	No. of Late Resorptions
G1 & 0	Mean	13.17	13.17	12.17	12.17	7.09	5.09	0.00	0.78	0.22
	±SD	2.95	2.95	2.72	2.72	2.31	1.81	0.00	1.04	0.42
	n	23	23	23	23	23	23	23	23	23
G2 & 50	Mean	12.58	12.58	12.00	12.00	6.13	5.88	0.00	0.54	0.04
	±SD	3.23	3.23	3.20	3.20	2.07	2.58	0.00	0.72	0.20
	n	24	24	24	24	24	24	24	24	24
G3 & 150	Mean	12.61	12.61	11.48	11.48	5.96	5.52	0.00	0.96	0.17
	±SD	1.80	1.80	2.09	2.09	2.23	2.00	0.00	1.02	0.49
	n	23	23	23	23	23	23	23	23	23
G4 & 500	Mean	12.67	12.42	11.54	11.54	5.71	5.83	0.00	0.17	0.33
	±SD	3.71	3.89	3.99	3.99	2.27	2.68	0.00	0.78	0.56
	n	24	24	24	24	24	24	24	24	24

 SD: Standard Deviation; n: No. of animals

TABLE 7.        SUMMARY OF MATERNAL DATA RECORD

Group & Dose (mg/kg body weight)		Pre-Implantation Loss (%)	Post-Implantation Loss (%)	Percent of Dead Fetus	Percent of Early Resorptions	Percent of Late Resorptions	Male / Female Sex ratio	Male Fetuses (%)	Female Fetuses (%)	Percent of Live Fetuses
G1 & 0	Mean	0.00	6.89	0.00	5.37	1.52	1.61	57.70	42.30	100.00
	±SD	0.00	8.65	0.00	7.13	2.97	0.86	13.15	13.15	0.00
	n	23	23	23	23	23	23	23	23	23
G2 & 50	Mean	0.00	4.57	0.00	4.31	0.26	1.41	50.81	49.19	100.00
	±SD	0.00	5.58	0.00	5.64	1.28	1.27	18.46	18.46	0.00
	n	24	24	24	24	24	24	24	24	24
G3 & 150	Mean	0.00	9.10	0.00	7.79	1.30	1.43	51.77	48.23	100.00
	±SD	0.00	8.79	0.00	8.18	3.86	1.33	15.21	15.21	0.00
	n	23	23	23	23	23	23	23	23	23
G4 & 500	Mean	1.92	7.75	0.00	4.81	2.94	1.22	54.00	51.56	100.00
	±SD	9.42	11.72	0.00	8.25	5.04	0.70	18.22	21.06	0.00
	n	24	24	24	24	24	24	24	24	24

G: Group; SD: Standard Deviation; n: Number of dams

TABLE 8.        SUMMARY OF ABSOLUTE THYROID ALONG WITH PARATHYROID WEIGHT (g) RECORD

Group & Dose (mg/kg body weight)		Thyroid along with parathyroid^#
G1 & 0	Mean	0.0246
	±SD	0.0045
	n	23
G2 & 50	Mean	0.0245
	±SD	0.0041
	n	24
G3 & 150	Mean	0.0238
	±SD	0.0022
	n	23
G4 & 500	Mean	0.0249
	±SD	0.0036
	n	24

SD: Standard Deviation; n: Number of animals; #: Weighed post fixation

TABLE 9.        SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT (%) RELATIVE TO TERMINAL BODY WEIGHT RECORD

Refer Appendix 9

Group & Dose (mg/kg body weight)		Terminal Body Weight (g)	Thyroid along with parathyroid
G1 & 0	Mean	404.13	0.0062
	±SD	37.83	0.0014
	n	23	23
G2 & 50	Mean	396.79	0.0062
	±SD	30.81	0.0011
	n	24	24
G3 & 150	Mean	394.24	0.0061
	±SD	29.76	0.0007
	n	23	23
G4 & 500	Mean	402.84	0.0062
	±SD	39.37	0.0011
	n	24	24

 SD: Standard Deviation; n: Number of animals

TABLE 10.        SUMMARY OF SERUM TRIIODOTHYRONINE (T3) LEVELS (ng/mL) RECORD

Group & Dose (mg/kg body weight)		Serum T3 Levels (ng/mL)
G1 & 0	Mean	2.823
	±SD	0.203
	n	23
G2 & 50	Mean	2.813
	±SD	0.204
	n	24
G3 &150	Mean	2.749
	±SD	0.236
	n	23
G4 & 500	Mean	2.469*
	±SD	0.254
	n	24

SD: Standard Deviation; n: Number of animals;*. The mean difference is significant at the 0.05 level

TABLE 11.        SUMMARY OF SERUM THYROXINE (T4) LEVELS (ng/mL) RECORD

Refer Appendix 11

Group & Dose (mg/kg body weight)		Serum T4 Levels (ng/mL)
G1 & 0	Mean	73.380
	±SD	6.397
	n	23
G2 & 50	Mean	71.373
	±SD	7.325
	n	24
G3 &150	Mean	67.687*
	±SD	5.189
	n	23
G4 & 500	Mean	64.964*
	±SD	4.666
	n	24

G: Group; SD: Standard Deviation; n: Number of dams;*. The mean difference is significant at the 0.05 level

TABLE 12.        SUMMARY OF SERUM THYROID STIMULATING HORMONE (TSH) LEVELS (µIU/mL) RECORD

Refer Appendix 12

Group & Dose (mg/kg body weight)		Serum TSH Levels (µIU/mL)
G1 & 0	Mean	5.717
	±SD	6.676
	n	23
G2 & 50	Mean	5.539
	±SD	3.888
	n	24
G3 & 150	Mean	5.171
	±SD	3.487
	n	23
G4 & 500	Mean	4.873
	±SD	2.785
	n	24

SD: Standard Deviation; n: Number of animals

TABLE 13.        SUMMARY OF FETAL WEIGHT (g) RECORD

Refer Appendix 13

Group & Dose (mg/kg body Weight)	Fetus Weight
		Mean Male Fetal Weight (g)	Mean Female Fetal Weight (g)
G1 & 0	Mean	4.35	3.99
	±SD	0.23	0.27
	n	23	23
G2 & 50	Mean	4.19	3.99
	±SD	0.28	0.29
	n	23	24
G3 & 150	Mean	4.07*	3.80*
	±SD	0.35	0.32
	n	23	23
G4 & 500	Mean	4.17	3.89
	±SD	0.19	0.24
	n	24	24

SD: Standard Deviation; n: Number of animals

*: The mean difference is significant at the 0.05 level

TABLE 14.        SUMMARY OF CROWN RUMP LENGTH (mm) RECORD                                                                                                                                         

                                                                                                          Refer Appendix 14

Group & Dose (mg/kg body weight)	CR Length (mm)
		Male	Female
G1 & 0	Mean	39.12	37.40
	±SD	2.05	2.66
	n	23	23
G2 & 50	Mean	38.49	37.33
	±SD	1.58	2.56
	n	23	24
G3 & 150	Mean	37.94	36.45
	±SD	2.67	2.82
	n	23	23
G4 & 500	Mean	38.33	36.80
	±SD	2.44	2.85
	n	24	24

SD: Standard Deviation; n: Number of animals

TABLE 15.        SUMMARY OF ANOGENITAL DISTANCE (AGD) RATIO RECORD

Refer Appendix 15

Group & Dose (mg/kg body weight)	AGD Ratio
		Mean Male AGD Ratio	Mean Female AGD Ratio
G1 & 0	Mean	2.55	1.39
	±SD	0.15	0.19
	n	23	23
G2 & 50	Mean	2.55	1.37
	±SD	0.10	0.10
	n	23	24
G3 & 150	Mean	2.54	1.28*
	±SD	0.13	0.14
	n	23	23
G4 & 500	Mean	2.60	1.36
	±SD	0.12	0.11
	n	24	24

SD: Standard Deviation; n: Number of animals;*:The mean difference is significant at the 0.05 level

TABLE 16.        SUMMARY RECORD OF FETAL EXTERNAL EXAMINATION

Refer Appendix16

Parameters	Group	G1	G2	G3	G4
	Dose (mg/kg body weight)	0	50	150	500
	Number of Dams	23	24	23	24
Number of fetuses for External Examination		280	288	264	277
Number of fetuses observed with No Abnormality Detected		277	285	261	273
Number of fetuses observed with Malformations		0	0	0	0
Number of fetuses observed with Variations		3	3*	3	4
VARIATIONS
HAEMORRHAGIC SPOT ON
Right Fore Limb (Unilateral)	No.	1 (1)	1 (1)	1 (1)	1 (1)
	%	0.36	0.35	0.38	0.36
Left Fore Limb (Unilateral)	No.	0 (0)	0 (0)	1 (1)	1 (1)
	%	0.00	0.00	0.38	0.36
Fore Limb (Bilateral)	No.	0 (0)	1 (1)	0 (0)	0 (0)
	%	0.00	0.35	0.00	0.00
Right Hind Limb (Unilateral)	No.	0 (0)	1 (1)	0 (0)	0 (0)
	%	0.00	0.35	0.00	0.00
Left Hind Limb (Unilateral)	No.	1 (1)	0 (0)	1 (1)	1 (1)
	%	0.36	0.00	0.38	0.36
Hind Limb (Bilateral)	No.	0 (0)	0 (0)	0 (0)	1 (1)
	%	0.00	0.00	0.00	0.36
Neck	No.	0 (0)	1 (1)	0 (0)	0 (0)
	%	0.00	0.35	0.00	0.00
Tail	No.	1 (1)	0 (0)	0 (0)	0 (0)
	%	0.36	0.00	0.00	0.00

Number of dams in parentheses

*: One fetus observed with haemorrhagic spot onbothright fore limb & neck region.

TABLE 17.        SUMMARY RECORD OF FETAL VISCERAL EXAMINATION

Parameters	Group	G1	G2	G3	G4
	Dose (mg/kg body weight)	0	50	150	500
	Number of Dams	23	24	23	24
Number of fetuses for visceral examination		132	138	127	133
Number of fetuses observed with No Abnormality Detected		129	131	123	131
Number of fetuses observed with Malformations		0	2	1	1
Number of fetuses observed with Variations		3	6	3	1
MALFORMATIONS
Head - Cleft palate (malformed)	No.	0 (0)	2 (2)	1 (1)	1 (1)
	%	0.00	1.45	0.79	0.75
VARIATIONS
Kidney - Renal pelvis Dilation - Unilateral (right)	No.	2 (2)	0 (0)	0 (0)	0 (0)
	%	1.5	0.00	0.00	0.00
Kidney - Pale colored - Unilateral (right)	No.	1 (1)	3 (3)	2 (2)	0 (0)
	%	0.76	2.17	1.57	0.00
Brain - Ventricles dilated	No.	0 (0)	2 (2)	0 (0)	0 (0)
	%	0.00	1.45	0.00	0.00
Liver, Adrenals, Kidneys: Pale colored	No.	0 (0)	1 (1)	0 (0)	0 (0)
	%	0.00	0.72	0.00	0.00
Kidney: Pale colored - Unilateral (left)	No.	0 (0)	0 (0)	1 (1)	1 (1)
	%	0.00	0.00	0.79	0.75

Number of dams in parentheses

 

		No. of fetuses with abnormality
% of Abnormality	:	------------------------------------------ X 100
		Total No. of fetuses examined

TABLE 18.        SUMMARY OF SKELETAL EXAMINATION OF FETUSES

Parameters	Group	G1	G2	G3	G4
	Dose (mg/kg body weight)	0	50	150	500
	Number of Dams	23	24	23	24
Number of fetuses for skeletal examination		148	150	137	144
Number of fetuses observed with No Abnormality Detected		117	116	109	119
Number of fetuses observed with Malformations		10	12	7	10
Number of fetuses observed with Variations		21	22	21	15
MALFORMATIONS
SKULL
Skull - Misshapen	No.	1 (1)	0 (0)	0 (0)	0 (0)
	%	0.68	0.0	0.0	0.0
STERNUM
Sternum No. 5 - Absent	No.	3 (3)	4 (4)	6 (6)	2 (2)
	%	2.01	2.67	4.38	1.39
Sternum No. 6 - Absent	No.	0 (0)	1 (1)	0 (0)	1 (1)
	%	0.00	0.67	0.00	0.72
Sternum No. 5 and 6 - Absent	No.	0 (0)	0 (0)	0 (0)	2 (2)
	%	0.00	0.00	0.00	1.39
FORE LIMB
Proximal phalanx No. 3 and 4 - Absent (Bilateral)	No.	6 (6)	7 (7)	1 (1)	5 (5)
	%	4.05	4.67	0.73	3.47

Number of dams in parentheses

                                                                           

		No. of fetuses with abnormality
% of Abnormality	:	------------------------------------------ X 100
		Total No. of fetuses examined

TABLE 18 (Contd..,). SUMMARY OF SKELETAL EXAMINATION OF FETUSES

Parameters	Group	G1	G2	G3	G4
	Dose (mg/kg body weight)	0	50	150	500
	Number of Dams	23	24	23	24
Number of fetuses for skeletal examination		148	150	137	144
Number of fetuses observed with No Abnormality Detected		117	116	109	119
Number of fetuses observed with Malformations		10	12	7	10
Number of fetuses observed with Variations		21	22	21	15
VARIATIONS
SKULL
Intra parietal bones - Poor Ossification	No.	1 (1)	3 (3)	4 (4)	0 (0)
	%	0.68	2.00	2.92	0.00
Supraoccipital bones - Poor Ossification	No.	2 (2)	2 (2)	2 (2)	1 (1)
	%	1.35	1.33	1.46	0.69
STERNUM
Sternum No. 5 - Poor Ossification	No.	1 (1)	2 (2)	0 (0)	0 (0)
	%	0.68	1.33	0.00	0.00
Sternum No. 5 - Incomplete Ossification	No.	2 (2)	0 (0)	0 (0)	0 (0)
	%	1.35	0.00	0.00	0.00
Sternum No. 5 - Bipartite Ossification	No.	2 (2)	0 (0)	1 (1)	0 (0)
	%	1.35	0.00	0.73	0.00
Sternum No. 6 - Poor Ossification	No.	2 (2)	1 (1)	0 (0)	0 (0)
	%	1.35	0.67	0.00	0.00
Sternum No. 6 - Incomplete Ossification	No.	0 (0)	6 (6)	6 (6)	2 (2)
	%	0.00	4.00	4.38	1.39
Sternum No. 5 and 6 - Incomplete Ossification	No.	2 (2)	0 (0)	0 (0)	0 (0)
	%	1.35	0.00	0.00	0.00

 Number of dams in parentheses

 

		No. of fetuses with abnormality
% of Abnormality	:	------------------------------------------ X 100
		Total No. of fetuses examined

TABLE 18 (Contd..,). SUMMARY OF SKELETAL EXAMINATION OF FETUSES

Refer Appendix 18

Parameters	Group	G1	G2	G3	G4
	Dose (mg/kg body weight)	0	50	150	500
	Number of Dams	23	24	23	24
Number of fetuses for skeletal examination		148	150	137	144
Number of fetuses observed with No Abnormality Detected		117	116	109	119
Number of fetuses observed with Malformations		10	12	7	10
Number of fetuses observed with Variations		21	22	21	15
VARIATIONS
RIBS
Rib No. 1 - Discontinuous (unilateral)	No.	1 (1)	0 (0)	0 (0)	0 (0)
	%	0.68	0.00	0.00	0.00
Rib No. 13 - Wavy	No.	3 (3)	2 (2)	0 (0)	4 (4)
	%	2.03	1.33	0.00	2.78
Rib No. 11,12 and 13 - Wavy	No.	0 (0)	0 (0)	1 (1)	1 (1)
	%	0.00	0.00	0.73	0.69
LUMBAR VERTEBRAE
Centrum No. 5 and 6 - Dumbbell shaped	No.	1 (1)	2 (2)	0 (0)	0 (0)
	%	0.68	1.33	0.00	0.00
Centrum No. 4 - Dumbbell shaped	No.	0 (0)	1 (1)	0 (0)	0 (0)
	%	0.00	0.67	0.00	0.00
SACRAL VERTEBRAE
Extra Ossification Site - Arch No.4	No.	2 (2)	0 (0)	4 (4)	2 (2)
	%	1.35	0.00	2.92	1.39
Centrum no. 4 - Poor Ossification	No.	1 (1)	1 (1)	0 (0)	0 (0)
	%	0.68	0.67	0.00	0.00
CAUDAL VERTEBRAE
Extra Ossification Site -  Arch No.2	No.	1 (1)	2 (2)	3 (3)	5 (5)
	%	0.68	1.33	2.19	3.47

 Number of dams in parentheses

 

		No. of fetuses with abnormality
% of Abnormality	:	------------------------------------------ X 100
		Total No. of fetuses examined

Applicant's summary and conclusion

Conclusions:

The Test item, 6-(Isononanoylamino) hexanoic acid, compound with 2, 2′, 2″-nitrilotriethanol (1:1) was administered via oral route at a dose volume of 10 mL/kg body weight to mated female rats from gestation day 5 (GD5) through gestation day 19 (GD19). The dose levels were 0 mg/kg, 50 mg/kg, 150 mg/kg and 500 mg/kg for the control (G1), low (G2), mid (G3) and high (G4) groups, respectively.

The results of the experiment support the conclusion that the NOAEL [No Observed Adverse Effect Level] for the test item, 6-(Isononanoylamino) hexanoic acid, compound with 2, 2′, 2″-nitrilotriethanol (1:1) for maternal and developmental toxicity toxicity is 500 mg/kg, the high dose, as there were no effects on maternal parameters as well as fetal parameters at all the tested dose groups.

Executive summary:

The objective of this study was to assess the effects of prenatal exposure of   6-(Isononanoylamino) hexanoic acid, compound with 2, 2′, 2″-nitrilotriethanol (1:1) on pregnant rats and on the developing fetus, including assessment of maternal effects as well as death, structural abnormalities, or altered growth in the fetus.

The test item,6-(Isononanoylamino) hexanoic acid, compound with  2, 2′, 2″-nitrilotriethanol (1:1), was administeredby the oral routeat 10 mL/kg body weight to mated and presumed-pregnant females from Gestation Day [GD] 5 to 19. For dams the end points of assessment were maternal death, maternal body weight and clinical signs of maternal toxicity. In the fetuses the end points of assessment were fetal death, structural variations and malformations or altered growth.

A total of 100 mated female Sprague Dawley rats were allocated to four groups. Each group consisted of 25 matedpresumed-pregnant female Sprague Dawley rats (Dams).6-(Isononanoylamino) hexanoic acid, compound with 2, 2′, 2″-nitrilotriethanol (1:1)was administered by the oral route in graduated doses at three dose levels:

Group G1: 0 mg/kg (Vehicle Control)

Group G2: 50 mg/kg (Low Dose)

Group G3: 150 mg/kg (Mid Dose)

Group G4: 500 mg/kg (High Dose)

All the animals were observed for clinical signs of toxicity once daily, mortalities twice daily during the experimental period, the body weight and feed consumption were recorded on 0, 3, 5, 8, 11, 14, 17, 19 and on 20(day of caesarean section). On the day of caesarean section, all the dams were observed for gravid uterus weight, uteri observations (no. of corpora lutea, no. of implantations, no. of resorptions and no of live and foetuses). The sex ratio, pre/post implantation loss per dam, percentage of male and female foetuses was calculated based on the uteri observations. The weight of thyroid along with the parathyroid was recorded post-fixation for all the group animals.

All the animals were euthanized on gestation Day 20 by exposing to CO₂and subjected to detailed gross pathological examination. The histopathology of thyroid along with parathyroid conducted for all the tested dose group animals.

A total number of 24, 23, 24 and 23 mated females were confirmed pregnant across groups yielding pregnancy rates of 96%, 92%, 96% and 92% at the time of caesarean section for the G1 (0 mg/kg), G2 (50 mg/kg), G3 (150 mg/kg) and G4 (500 mg/kg), respectively.

No clinical signs of toxicity and no mortalitieswere noted during the experimental period at all the tested dose group animals.No treatment related effects were observed on gestational body weight, body weight gain and corrected body weight across the dose groups when compared with controls. There were no treatment related differences in feed consumption across the dose groups during gestation period.

No treatment related changes were noted ingravid uterus weight, uteri observations (no. of corpora lutea, no. of implantations, no. of resorptions and no of live and foetuses), sex ratio, pre/post implantation loss per dam, percentage of male and female foetuses and weight of thyroid along with the parathyroid wat all the tested dose groups.

No gross pathological findings were reported in all the tested dose groups during conductance of the necropsy.

The histopathology of thyroid along with parathyroid conducted for all the dose group dams did not reveal any treatment related histopathological findings during conduct of the histopathological examination. Presence of ultimobranchial cysts in G1 (2 incidences), G2 (1 incidence), G3 (4 incidences) and G4 (3 incidences); ectopic thymus in G1 (1 incidence) and G4 (3 incidences) were congenital lesions and it does not have toxicological significance.

For the maternal toxicity assessment, the results of the experiment support that there were no treatment related effects on maternal body weight, feed consumption, forthe parameters which assess gestational integrity end points, e.g.mean gravid uterus weight, number of corpora lutea, number of implantations, litter size, number of live fetuses, number of dead fetuses,mean number of early resorptions per dam and percentage of implantation losses per damacross all the groups.

The gravid uterus was collected by hysterectomy and fetuses were removed by caesarean section. The fetuses were observed for fetal weights, crown rump length, ano-genital distance, observation of reproductive tract, comparison of external fetal sex with internal gonads. Examination of fetuses forexternal, soft tissue, and skeletal anomalieswas performed.

Notreatment relatedeffects on fetal weight, crown-rump length, fetal ano-genital distance ratio and comparison of external fetal sex with internal gonads were noted at all the tested dose groups.

No treatment related gross external abnormalities were noted within any group after external examination of fetuses. The noted findings of haemorrhagic spots on the are common findings for fetuses of this species and strain.

No treatment-related abnormalities were observed during visceral examinations of fetuses at any dose. The noted findings of renal pelvic dilation, pale colored kidneys, and pale colored adrenals are anatomical variations which are common findings for fetuses of this species and strain. The observations were not dose dependent, nor were the severity of the anomaly increased with dose. This result supports the conclusion that the findings are incidental and that the test item did not produce an adverse effect on the soft tissues during fetal development.

No treatment-related abnormalities were observed during skeletal examinations of fetuses at any dose. Split thoracic/lumbar vertebral centrum; absence of proximal phalanges no. 3 and 4; absence of sternum no. 5 and 6 were recorded as malformations. Poorly ossified sternum no. 5 and 6, sacral vertebrae centrum, dumbbell shaped thoracic/lumbar/sacral vertebrae, extra ossification site on caudal vertebrae and wavy ribs were recorded as variations. The noted anomalies are common findings for fetuses of this species and strain. They did not occur in a dose-dependent pattern, nor was the severity of the anomalies increased with dose. This result supports the conclusion that the findings are incidental and that the test item, did not produce an adverse effect on the skeletal tissues during fetal development. 

For the developmental toxicity assessment, the results of the experiment support that there were notreatment relatedeffects on the parameters which assess fetal development, i.e. fetal weight, crown-rump length, fetal ano-genital distance ratio and incidences of external, soft tissue or skeletal anomalies.

The results of the experiment support the conclusion that the NOAEL [No Observed Adverse Effect Level] for the test item,6-(Isononanoylamino) hexanoic acid, compound with 2, 2′, 2″-nitrilotriethanol (1:1)for maternal and developmental toxicitywas 500 mg/kg, the high dose, as there were no effects on maternal parameters as well as fetal parameters at all the tested dose group