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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31-May-2017 to 19-Oct-2017
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Denomination:Hexadecyl 3,5-bis-tert-butyl-4-hydroxybenzoate.

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, Domaine des Oncins, 69210 Saint Germain sur l'Arbresle, France.
- Age at study initiation: 10 to 13 weeks.
- Weight at study initiation: 231 to 291 g
- Housing: One air-conditioned room in a barrier protected unit (building K4). Animals were singly housed in plastic cages, in compliance with European Regulations (Directive 2010/63/EU).
- Diet (e.g. ad libitum): Rat pelleted complete diet ad libitum (Diet reference A04C-10) sterilised by irradiation and analysed for a predefined list of chemical and bacteriological contaminants. Each batch of diet is supplied with a certificate of analysis which is verified and authorized for release by a veterinarian.
- Water (e.g. ad libitum): Softened and filtered (0.2 µm) mains drinking water was available ad libitum (via bottles). Water is analysed twice a year for chemical and bacterial contaminants by Laboratoire Santé Environnement Hygiène de Lyon, France.
-Bedding: Dust-free sawdust (SDS/Dietex, Argenteuil, France) made from spruce tree wood, analysed at least twice a year for chemical and bacterial contaminants.
-Enrichment: A small amount (handful) of shredded paper (SDS/Dietex) was provided as enrichment for all animals. Furthermore, animals had free access to a wooden gnaw block (Aspen bricks, Le comptoir des sciures, France).

- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 3 °C (target range).
- Humidity (%): > 35 % (target).
- Air changes (per hr): At least 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light (artificial)/12 hours dark.

IN-LIFE DATES: From:2017-07-06 To: 2017-07-21

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was prepared as a suspension in the vehicle at concentrations of 50, 100 and 200 mg/mL for the DRF and main phases, according to Standard Operating Procedures of the Test Facility.
Correction factor: None.
Frequency of preparation: Weekly.
Storage of formulations: At ambient temperature or refrigerated (between +2 and +8 °C), protected from light.

VEHICLE
- Justification for use and choice of vehicle :
- Concentration in vehicle: 0, 50, 100 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): MKCB2122V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Method description
¤ HPLC (pump, autosampler, detector): 1100 Series Agilent Technologies HPLC
¤ Column: Zorbax TMS 250 mm x 4.6 mm, 5 µm, référence : 880952-710
¤ Precolumn: NA ¤ Injection volume: 10 µL
¤ Oven temperature: 40 °C ¤ Needle wash (Acetonitrile)
¤ Run time: 15 minutes ¤ Autosampler at ambient temperature, not controlled
¤ UV Detection: 254 nm ¤ Retention time: around 9 minutes
¤ Elution isocratic: ¤ Flow rate: 2 mL/minute
- Mobile Phase: 70% (76 % Acetonitrile/ 24 % ultra pure water, 0.001M KH2PO4 , pH3.0) / 30% of Acetonitrile

¤ Test item used for calibration standard and check standard:
- Denomination: Hexadecyl 3,5-bis-tert-butyl-4-hydroxybenzoate
- Other denomination: CYASORB® (UV-2908) or CYASORB® (UV-2908) Light Stabilizer or UV-2908
- Batch number: WP6061904 Retest date: 19 June 2018
- correction factor: none (considerate at 100 %).
¤ Calibration range and Quality Control (QC):
20, 50, 100, 150 and 200 µg/mL prepared from two independant standard solutions
- Standard solutions was prepared at 400 µg/mL in dilution solvent
- Dilution solvent: Acetonitrile
- Dilution factors: 1/20, 1/8, 1/4, 3/8, 1/2 respectively.
¤ Formulation concentration range validated: 1 to 200 mg/mL
in: Propylene Glycol
¤ Electronic systems:
Microsoft® Office Excel 2010, Provantis 9 (Data acquisition, Dispensary)
Chemstore B.02.01 and ChemStation for LC A.08.04 (HPLC analysis)

¤ Analysis conformity:
- Calibration solutions (SE): recovery within 95-105 % of the exact concentrations (except for LLOQ samples 90-110 %): "
Pass"
- QC solutions: recovery within 95-105 % of the exact concentrations (except for LLOQ QC samples 90-110 %). One QC sample may be outside the acceptance criteria: "
Pass"
Formula
1- Linearity of the calibration curves: y = ax+b
y = test item peak area (mAUs)
x = concentrations of the test item calibration solution (µg/mL)
2- "Standard solution conformity : Deviation between the Calibration standard solutions (SS Cal)
and Quality Control standard solutions (SS QC)"
" SS Cal area
Deviation (%) = [——————————— - 1] x 100
SS QC area"
3- Analysis conformity, The recovery of the back-calculated concentration of SE or SQC
" Individual back calculated concentration
Recovery (%) = ————————————————————— x 100
nominal concentration
"
4- Concentrations of the formulation samples (suspension):
" y - b V x dilution factor x d
Concentration (mg/mL) = ——— x ——————————
a sample weight x 1000 " "with V:volume of the
volumetric flask used
d : formulation density
Sample weight in g"
5- Deviation from the nominal concentration of the formulations:
" mean of experimental concentration – nominal concentration
Deviation (%) = ————————————————————————— x 100
nominal concentration"

Details on mating procedure:
- Impregnation procedure: purchased timed pregnant on day 0 of gestation
Duration of treatment / exposure:
From day 6 (G6), implantation, to day 20 (G20) of gestation inclusive.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22 animals per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on DRF study results

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at least twice daily (including weekends and public holidays).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on days 0, 6, 9, 12, 15, 18 and 21 of gestation.

BODY WEIGHT: Yes
- Time schedule for examinations: on days 0, 6, 9, 12, 15, 18 and 21 of gestation.

FOOD CONSUMPTION : Yes
- Individual food consumption was measured for the periods (days) 0 to 6, 6 to 9, 9 to 12,12 to 15, 15 to 18 and 18 to 21 during gestation.

WATER CONSUMPTION : No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: ovaries, uterus.placentae
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:
Statistical analyses were performed by the Provantis data acquisition system, where appropriate, as follows:
The best transformation for the data (none, log or rank) was determined depending upon
- the kurtosis of the data
- the probability of the Bartlett's test for homogeneity of the variances and
- an assessment of whether the size of the groups were approximately equal or not.
Non- or log-transformed data were analysed by parametric methods.
Rank transformed data were analysed using non-parametric methods.
Data were then analysed to test for a dose-related trend to detect the lowest dose at which there was a significant effect, based on the Williams test for parametric data or the Shirley's test for non-parametric data.
Homogeneity of means was assessed by analysis of variances (ANOVA) for parametric data or Kruskal-Wallis test for non parametric data.
If no trend was found and means were not homogeneous, the data were analysed by parametric or non-parametric Dunnett's test to look for significant differences from the control group.
Selected incidence data were analysed using the Provantis data acquisition system and/or a SAS software package. A chi2 test was used for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 was significant.
Microsoft Excel® (version 2003 or higher) was employed to present certain results.
Historical control data:
Yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item-related clinical sign in any group.
Incidental clinical signs were noted such as localized hairloss, scabs and chromodacryorrhea.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weight and mean body weight gain were essentially comparable in all groups (including the control group).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no test item-related effect on mean food consumption in any group during the dosing period.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There was a slightly lower mean gravid uterus weight in the 250 and 1000 mg/kg/day group (68.2 and 67.9g, respectively) compared with the control (71.4g). However this effect was not dose related and the values were only slightly below the historical control data range (70.4 – 72.4g). This effect was therefore considered incidental.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There was no test item-related macroscopic finding in any group.
Incidental findings including sores/crusts or alopecia were noted sporadically. All were correlated with previous clinical observations noted in 2 females in each of the treated groups.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The mean numbers of corpora luteas and implantation sites in the treated groups were slightly higher than those in the control group but were inside the historical control data range.
The percentage of pre-implantation loss was high in all groups (including the control) compared to the historical control data (7.4% with a range of 0 – 13.3%). However, the values were exacerbated by one female in each of the control (no.121), 250 (no.130) and 1000 mg/kg/day (no.187) and two females at 500 mg/kg/day (nos.164 and 166) with a percentage of loss above 42%). The pre-implantation loss data reflected the incidental variations.

There was no test item-related effect on post-implantation loss in any group.
The mean live litter size was comparable in all groups.

Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
The number of early resorptions for each control female ranged between 0 and 3. Incidentally, one female at 250 (no.135) and two females in each of the 500 and 1000 mg/kg/day groups had 5 (nos. 154 and 156) or 4 (nos. 180 and 185) early resorptions, respectively.
The percentage of post-implantation loss was consequently slightly higher in all treated groups than in the control group.
In excluding the females with more than 3 early resorptions, the percentage of post-implantation loss in all treated groups (9.9, 10.7 and 11.4% in the 250, 500 and 1000 mg/kg/day groups, respectively) was comparable to that of control (8.2%) and within the historical control data range (1.2 to 12.9).
Dead fetuses:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: There was no evidence of maternal toxicity in this study at the highest dose level tested

Results (fetuses)

Fetal body weight changes:
no effects observed
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
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A subcutaneous hemorrhage was noted as anomaly for the small foetus no.11 (2.38g) from dam no.124 given 250 mg/kg/day.
These findings are part of the background of changes for the strain of rat and were considered incidental due to their isolated nature.
There were 2 foetuses malformed from as many litters in the 500 and 1000 mg/kg/day group compared with none in other groups.
Both foetuses had one malrotated hindlimb (foetus no.3, 5.09g, from dam no.151 and foetus no.9, 5.31g, from dam no.169).
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Foetus no.11 from dam no.124 with multiple visceral abnormalities also had sternebrae, vertebrae and sacrum multiple abnormalities
The incidence of other skeletal anomalies and variations did not suggest any association with the test item.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were 2 other malformed foetuses from as many litters at the visceral examination.
At 500 mg/kg/day, one foetus had a situs inversus (foetus no.6, 4.57g, dam no.151).
At 250 mg/kg/day, the small foetus with a subcutaneous hemorrhage at external examination also had multiple urogenital and great vessels abnormalities (foetus no.11, 2.38g, dam no.124).
The incidence of other visceral anomalies and variations did not suggest any association with the test item.

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There was no evidence of developmental toxicity in this study at the highest dose level tested

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Summary of malformations – Individual descriptions:

Dose level (mg/kg/day)

Female number

Foetus number(s)

Malformation(s)#

250

124

11

Multiple urogenital abnormalities: testes malpositioned cranially, left epididymis and left kidney absent with dilated renal pelvis of right kidney and dilated and convoluted right ureter.

Multiple great vessels abnormalities: interrupted aortic arch terminating with left carotid artery, descending aorta arising from pulmonary trunk with left subclavian artery arising from decending aorta.

Multiple abnormalities of the vertebrae: gross disruption from 1stcervical to 13ththoracic with fused and absent ribs; scoliosis.

Multiple abnormalities of the sternebrae: fused and misshapen with numbering not possible.

Sacral, Multiple abnormalities of the sacrum: 2ndcentrum small and hemicentric; scoliosis.

500

151

3

Malrotatedright hindlimb.

6

Situs inversus (thoracic and abdominal).

1000

169

9

Malrotated left hindlimb.

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of the study, oral (gavage) administration of Hexadecyl 3,5-bis-tert-butyl-4-hydroxybenzoate to the pregnant Wistar rat from implantation through to the day before caesarean section at 250, 500 or 1000 mg/kg/day was well tolerated with no evidence of maternal toxicity in any group.
Similarly, there was no evidence of developmental toxicity in any group.
The No Observed Effect Level (NOEL) for both maternal and developmental toxicity was the limit dose of 1000 mg/kg/day.
Executive summary:

Objectives

The objective of this study wastodetermine the potential of the test item, Hexadecyl 3,5-bis-tert-butyl-4-hydroxybenzoate,to induce developmental toxicity after maternal exposure during the critical period of organogenesis, to characterize maternal toxicity at the exposure levels tested and to determine the NOAEL (no observed-adverse-effect level) for maternal toxicity and developmental toxicity in rats.

 

Procedures

Hexadecyl 3,5-bis-tert-butyl-4-hydroxybenzoate was administered by daily gavage at dose levels of 250, 500 and 1000 mg/kg/day to groups of 22 mated Wistar rats from days 6, implantation, to 20 of gestation inclusive. A control group received a similar volume (5 mL/kg) of the vehicle (propylene glycol).

Parameters monitored included morbidity/mortality, clinical signs, body weight and food consumption.

The females were submitted to a caesarean examination on day 21 of gestation and litter parameters were recorded. At necropsy, the females were examined macroscopically and the gravid uterus was weighed.All live foetuses were weighed, sexed and examined for external abnormalities. Half of the foetuses were examined internally prior to processing for skeletal examination. The remaining foetuses were preserved for fixed-visceral examinationof the head onlyby the modified Wilson-Barrow technique.

Results

Accuracy and homogeneity of formulations were demonstrated by the analyses.

Maternal findings

There was no unscheduled death in any group.

There were no test item-related clinical changes.

There were no test item-related effects on mean body weight gain and food consumption.

There were no treatment-related macroscopic findings or effect on mean gravid uterus weight in any group.

There were 22, 21, 22 and 21 pregnant females at terminal caesarean in the control, 250, 500 and 1000 mg/kg/day groups, respectively, all of which had viable foetuses.

Developmental findings

There was no test item-related effect on embryo-foetal survival, foetal weight or foetal sex ratio in any group.

There was no test item-related morphological change amongst the foetuses in any group.

There was one litter in each of the treated groups containing malformed foetus(es):

·      One foetus had multiple visceral and skeletal abnormalities in the 250 mg/kg/day.

·      One foetus in the 500 mg/kg/day group had a situs inversus.

·      One foetus in each of the 500 and 1000 mg/kg/day groups had a malrotated hindlimb.

Conclusion

Under the experimental conditions of the study, oral (gavage) administration of Hexadecyl 3,5-bis-tert-butyl-4-hydroxybenzoate to the pregnant Wistar rat from implantation through to the day before caesarean section at 250, 500 or 1000 mg/kg/day was well tolerated with no evidence of maternal toxicity in any group.

Similarly, there was no evidence of developmental toxicity in any group.

The No Observed Effect Level (NOEL) for both maternal and developmental toxicity was the limit dose of 1000 mg/kg/day.