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Developmental toxicity / teratogenicity

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Administrative data

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

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Beige lumps
Details on test material:
Study Sponsor (Name and Address): Clariant India Private Ltd, BU PIGMENTS, Product Stewardship India, Reliable Tech Park, Gut No. 31,
Village Elthan, Thane-Belapur Road, Airoli, Navi Mumbai 400 708, India

Batch Manufactured by: Arizona Chemical

Test item: Resin 835A

Chemical name : Reaction mass of Rosin, hydrogenated and [1R-(1a,4ab,10aa)]-1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-dimethylphenanthren-1-carboxylic acid

Batch No.: K49E09-03119

Manufactured Date: 08.03.2012

Expiry Date: 11.04.2017

Purity as per Certificate of Analysis::99.9 % (w/w)

Physical Appearance: Beige lumps

Storage Conditions: Ambient (+15 to +25ºC)

Certificate of Analysis: AZ 741/Tox99, dated 11. April 2012

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Vivo Bio Tech Ltd, Sy # 349/A, Pregnapur-502311, Gajwel Mandal, Medak District, Telangana

- Age at study initiation: 14 to 15 weeks
- Weight at study initiation:
Mean body weight Body weight range
G1 : 214.81 ± 15.14 190.17 to 242.11 g
G2 : 214.98 ± 16.29 189.40 to 254.11 g
G3 : 214.74 ± 15.53 189.13 to 248.21 g
G4 : 214.77 ± 16.16 188.17 to 241.53 g


- Housing:
Rats were housed in standard polysulfone rat cages (size: Length 425 mm x Breadth 266 mm x Height 185 mm) with stainless steel top grill having facilities for pellet food and drinking water in polycarbonate bottle with stainless steel sipper tube.

i. Pre mating / Acclimatization: Two rats of the same sex per cage were housed.
ii. Mating: Female rats were cohabited with males in a 1:1 ratio in same cage.
iii. Post-mating / Treatment: After mating confirmation, females were housed individually.

- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet (Certified) manufactured by Harlan Laboratories, P.O.Box 44220, Madison Wi 53744-4220, was provided ad libitum to the animals.

- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.

- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23 °C
- Humidity (%): 65 – 67 %,
- Air changes (per hr): 12 to 15
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle.

IN-LIFE DATES: From: 21 April 2016 To: 19 May 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.1% Sodium carboxymethyl cellulose (high viscosity) with 0.01% Tween 80 in Milli-Q water
Details on exposure:
DOSE FORMULATION PREPARATION:
The dose formulations were prepared in an interval of 1 to 3 days and used within the established stability period.

Required quantities of the test item was weighed on an aluminium paper and then was transferred to the mortar. The test item was ground to a fine powder using pestle; small volume of vehicle [0.1% Sodium carboxymethyl cellulose (high viscosity) with 0.01% Tween 80 in Milli-Q water)] was added gradually to the mortar with continuous trituration until a uniform suspension was obtained. The mixture was quantitatively transferred into the pre-calibrated beaker. Further, a small volume of vehicle was added to the mortar and washed with vehicle, all the rinsing was quantitatively transferred into the pre-calibrated beaker. The final volume was made up with vehicle to the desired volume to get the desired concentrations.

The homogeneity of the Resin 835A formulation during treatment/sampling was maintained by constant stirring using a magnetic stirrer.

Pre-calibration of the beaker to desired volume: Milli-Q water was measured in a graduated cylinder to the final volume (for example 150 mL). The measured water was transferred into a clean beaker (to be pre-calibrated) and upper and lower meniscus of water was marked on the beaker, the water was discarded and the beaker was dried. The upper meniscus mark was used to make up to the volume while preparing the dose formulations.

For vehicle control groups, vehicle [0.1% Sodium carboxymethyl cellulose (high viscosity) with 0.01% Tween 80 in Milli-Q water)] was administered.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulations were analyzed for active ingredient concentration (a.i.) and homogeneity in duplicate sets at the initiation of treatment and at termination of treatment period.

Formulations were considered acceptable when the overall mean result (calculated using all the 6 replicate values) of all the layers and mean of each layers was within ± 15.0 % of the claimed concentration and the relative standard deviation (% RSD, calculated using all the 6 replicate values) of assay of top, middle and bottom layers was equal to or less than 10.0 %.
Details on mating procedure:
During the mating period, female rats were cohabited with males in a 1:1 ratio. Presence of sperm in a vaginal smear or observation of vaginal plug in the morning, the animal was considered to be mated. This day was considered as Day ‘0’ of gestation.

The mated female rats obtained each day were assigned to the treatment groups and vehicle control groups by body weight stratification. This procedure was continued till the required numbers of Day 0 mated females were obtained (24 per group).
Duration of treatment / exposure:
The dose formulations of Resin 835A were administered orally by gavage using disposable plastic syringe attached with a metal feeding/intubation cannula to rats of low dose (G2), mid dose (G3) and high dose (G4) groups once daily from GD 5 to GD19 of presumed gestation, at approximately the same time each day (varying by± 2 hours).

The dose volume ( at 10 mL/kg) to be administered was calculated based on the body weight of individual animals on first day of treatment
(on GD 5) and was adjusted according to the most recently recorded body weights recorded till GD 19. The animals in the vehicle control group (G1) were handled in an identical manner to the treatment group and were administered vehicle only.
Frequency of treatment:
Gestation days: 5 to 19, once daily
Duration of test:
21 April 2016 to 18 August 2016
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Group G1
Dose / conc.:
50 mg/kg bw/day
Remarks:
Group G2
Dose / conc.:
250 mg/kg bw/day
Remarks:
Group G3
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Group G4
No. of animals per sex per dose:
24 day '0' pregnant rats per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:

Based on available toxicity data doses were selected with the aim to induce

- some developmental and/or maternal toxicity but no death or severe suffering at the highest dose
- minimal observable toxic effects at the intermediate dose
- no evidence of either maternal or developmental toxicity at the lowest dose



- Rationale for animal assignment (if not random):

The mated female rats were distributed to different groups by body weight stratification. The distribution of the Day ‘0’ pregnant rats obtained on each day was as follows: Based on the body weight, body weights were arranged in the ascending order. These rats were then assigned to the groups starting from control to treatment groups and then in the reverse order of dose groups to control.

Examinations

Maternal examinations:
Observations for clinical signs were performed twice a day - pre dose and post dose (within 1-2 hours of administration) during treatment days and once on non-treatment days.

Each rat was observed twice daily for morbidity and mortality i.e., once in the morning and once in the afternoon. Based on the assessment, as there were toxic signs of concern, the observation was carried out twice during weekends and public holidays.
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 tests were used:

The data on maternal body weight, body weight change, gravid uterine weight, corrected body weight on gestation day 20 (carcass weight), corrected body weight gain, maternal food consumption, number of corpora lutea, number of implantations, total number of fetuses, male and female fetus number and weight including combined sex was analyzed using ANOVA model, after testing for homogeneity for intra group variance using Levene’s test. When intra group variances were heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed when the group differences are found significant.

Incidences of pre-implantation loss, post implantation loss, Number of early and late resorptions were analyzed using Kruskal Wallis.

Overall percentage of minor external, visceral and skeletal malformations, Sex ratio and number of dams with any resorptions were analyzed using 2 X 2 Contingency Table.

Statistically significant differences (p < 0.05), indicated by the aforementioned tests were designated by symbol ‘*’ throughout the report

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
body weights were unaffected, except for a significant lower mean body weight on GD 11 (-6.3%) at 1000 mg/kg/day as compared to vehicle control group.

body weight gains at 50 mg/kg/day were comparable to vehicle control
body weight gains at 250 mg/kg/day were significantly reduced during GD 5-8 (-94.1%) as compared to vehicle control
body weight gains at 1000 mg/kg/day were significantly reduced during GD 5-8 (-254.6%), and during treatment period 5-20 (-15.7%), and significantly increased during GD 17-20 (+47.7%) as compared to vehicle control
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
food consumption at 50 mg/kg/day was comparable to vehicle control group
food consumption at 250 mg/kg/day was significantly reduced during GD 5-8 (-15.7%) as compared to vehicle control group
food consumption at 1000 mg/kg/day was significantly reduced during GD 5-8 (-45.9%), GD 8-11 (-29.1%), GD 11-14 (-15.1 %), during treatment period 5-20 (-17.3%) and for the entire gestation period 0-20 (-13.4%)
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
At the doses tested there were no changes in uterine weight
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological findings in any of the dams from the treated groups and control, including gross evaluation of placenta.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
pre- and post-implantation losses were comparable to the vehicle control group
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
early and late resorptions were comparable to the vehicle control group
Dead fetuses:
no effects observed
Description (incidence and severity):
G1: 0
G2: 0
G3: 0
G4: 0
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
A total of 24 sperm positive females were included in the vehicle control and each of the three treatment groups. The number of rats sacrificed at term was 24 in all the groups and the number of non-pregnant rats was 3, 0, 1 and 2 at 0, 50, 250 and 1000 mg/kg/day, respectively.
Other effects:
not specified

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: decrease in food consumption associated with lower body weights during the initial phase of tretament (Day 5 -8) at a dose of 250 mg/kg bw/day is considered a treatment-related transient non-adverse change
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: decrease in food consumption associated with lower body weights during the initial phase of tretament (Day 5 -8) at a dose of 250 mg/kg bw/day is considered a treatment-related transient non-adverse change

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
at 1000 mg/kg/day there was a significant decrease in mean fetal weights for combined sex (-7.9%), male (-8.9%) and female (-7.3%)
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): at 1000 mg/kg/day there was a significant decrease in mean fetal weights for combined sex (-7.9%), male (-8.9%) and female (-7.3%)
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
mean litter size is statistically comparable to the vehicle control group
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related malformations were observed at 50 mg/kg/day. Incidence of minor anomaly namely, small fetus was observed in 2/260 and 1/246 fetuses at 250 and 1000 mg/kg/day doses, respectively which were comparable to vehicle control group (0/246 fetuses) and were hence considered not to be of any significance.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Minor Anomalies:

at 1000 mg/kg/day: significant increase in instances of Extra rib No. 14 (Inc. 9), and Accessory rib No.14 (Inc. 8), and Rudimentary rib No.14 (Inc. 68) as compared to vehicle control group (Inc. 1/0/26)
at 250 mg/kg/day: significant increase in instances of Rudimentary rib No.14 as compared to vehicle control group

The incidence of Rudimentary rib No.14 (Inc. 49) at 250 mg/kg/day was within the historical control range.

The findings of increased instances of Extra rib No. 14, Accessory rib No.14 and Rudimentary rib No.14 at 1000 mg/kg/day were considered as treatment-related and were attributed to maternal stress due to treatment as evidenced by reduction in maternal body weight / body weight gain, and food consumption apparent at doses of >= 250 mg/kg/day.
Visceral malformations:
no effects observed
Description (incidence and severity):
no treatment-related changes were observed in the visceral examination of fetuses from dams treated up to 1000 mg/kg/day
Other effects:
not specified

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
Remarks on result:
other: The incidences of Rudimentary rib No.14 at 250 mg/kg/day was within the historical control range.

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: rib
Description (incidence and severity):
Minor Anomaly at 1000 mg/kg/day: significant increase in instances of Extra rib No. 14 (Inc. 9), and Accessory rib No.14 (Inc. 8), and Rudimentary rib No.14 (Inc. 68) as compared to vehicle control group (Inc. 1/0/26)

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects

Any other information on results incl. tables

Defnitions:

Extra rib: ½ or greater than the length of 13th rib in rats

Accessory rib: more than 1/3rd the length of 13th rib in rats

Rudimentary rib: less than 1/3rd the length of 13th rib in rats

Applicant's summary and conclusion

Conclusions:
Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for Maternal toxicity and Fetal developmental toxicity is 250 mg/kg/day in Wistar rats when Resin 835A was administered orally by gavage during GD 5 to 19 under the test conditions and doses employed in this study due to

a) Treatment-related significant reduction in maternal body weight gains and food consumption at 1000 mg/kg/day
b) Significant decrease in fetal body weights and increase in incidence of minor anomalies which are Extra, Accessory and Rudimentary rib no.14 at 1000 mg/kg/day
Executive summary:

The objective of this study was to evaluate the embryo-fetal developmental toxicity of test item Resin 835A when administered to pregnant Wistar rats by oral route during gestation days (GD) 5 to GD19. The results of this study helped to establish the No Observed Adverse Effect Level (NOAEL) of the test item.

 

Ninety six presumed pregnant Wistar rats were assigned to four groups by body weight stratification (Group 1 to Group 4) and each group (G1: control, G2: low dose, G3: mid dose and G4: high dose) consisted of 24 presumed pregnant rats (gestation day 0). Day `0' of gestation for each individual female rat in the study was considered as the day on which vaginal smear was found positive for sperm.

 

The test item, Resin 835A was suspended in 0.1% Sodium carboxymethly cellulose (high viscosity) with 0.01% Tween 80 in Milli-Q water and administered orally (by gavage) to presumed pregnant rats once daily from GD 5 to 19 at the dose levels of 50, 250 and 1000 mg/kg/day for low (G2), mid (G3) and high (G4) dose group rats, respectively. Rats in the vehicle control (G1) group received the vehicle (0.1% Sodium carboxymethly cellulose (high viscosity) with 0.01% Tween 80 in Milli-Q water) alone. A constant dose volume of 10 mL/kg body weight was administered to all groups.

 

The identity of the test item was provided by the study sponsor by a Certificate of Analysis. The stability and homogeneity of the test item in the vehicle was established under Study No. G11397 at concentrations of 3 and 100 mg/mL and the results of the study indicated that the test item was found to be stable and resuspendable in the vehicle for 4 days at room temperature. During the conduct of the experiment, homogeneity and active ingredient analysis was carried out from the dose formulation samples collected at the initiation and termination of treatment. The results of analysis of formulations were within the acceptable limits.

 

The mated females were observed twice daily for clinical signs (during treatment period), mortality and morbidity. Body weights were recorded on GD 0, 3, 5, 8, 11, 14, 17 and 20. About 200 g (food input) was provided on GD ‘0’. The food left over was recorded and replenished to a known weight on GD 3, 5, 8, 11, 14 and 17. The food left over was also recorded on Day 20 of presumed gestation. The intermittent body weight gain and food intake was calculated and presented for rats found pregnant at caesarean section.

 

Caesarean section was performed for all rats on GD 20 and dams were examined for gross pathological changes. The uterus from all the animal were removed (by laparotomy) and the contents were examined. The uteri were weighed and examined for the number of implantation sites, early and late resorptions, and number of live and dead fetuses. The number of corpora lutea was counted on each ovary. All the fetuses were sexed, weighed and examined for external malformations. Approximately half the number of fetuses from each dam was examined for visceral malformations and the remaining half was evaluated for skeletal malformations.

 

Results of the study are presented below:

·       There were no mortality or clinical signs. At terminal sacrifice there were no gross pathological changes in any of the dams at the dose levels tested.

·       There was treatment-related significant reduction in maternal body weights gains during the treatment period GD 5 to 20 (-15.7%) and food consumption during the treatment period GD 5 to 20 (-17.3%) and throughout gestation period 0 to 20 (-13.4 %) at 1000 mg/kg/day as compared to vehicle control group.

·       At the doses tested there were no changes in maternal parameters such as the number of corpora lutea, uterine weight, implantations, early and late resorptions. Gross evaluation of placenta revealed no treatment related findings.

·       The litter data parameters such as the total number of fetuses, number of live fetuses and sex ratio were statistically comparable to vehicle control group at all the doses tested. At 1000 mg/kg/day, the male and female fetal weights (-8.9% and -7.3%, respectively) were significantly lower and were considered to be treatment-related.

·       Fetal external and visceral observations revealed no signs of fetal malformations or developmental toxicity at all the doses tested.

·       Fetal skeletal examination revealed significant increase in minor anomalies such as Extra, Accessory and Rudimentary rib No.14 at 1000 mg/kg/day and this finding was attributed to maternal stress due to treatment as evidenced by decrease in maternal body weight / body weight gain, and food consumption.

·       Visceral and skeletal examinations revealed no signs of teratogenicity in any of the tested dose levels up to 1000 mg/kg/day.

 

Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for Maternal toxicity and Fetal developmental toxicity is 250 mg/kg/day in Wistar rats when Resin 835A was administered orally by gavage during GD 5 to 19 under the test conditions and doses employed in this study due to

a) Treatment-related significant reduction in maternal body weight gains and food consumption at 1000 mg/kg/day

b) Significant decrease in fetal body weights and increase in incidence of minor anomalies, which are Extra, Accessory and Rudimentary rib no.14 at 1000 mg/kg/day