Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 7, 1980 through September 12, 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Insufficient documentation of experimental methods and examination procedures. In general, this study was performed according to the SOPs, which are in conformity with the FDA GLP Regulations. Deviations were limited, listed below, and are believed not to impact the reliability of the test result.
Qualifier:
according to
Guideline:
other: Breeding/Reproductive Study
Deviations:
yes
Remarks:
See Below
Principles of method if other than guideline:
a. The first amendment was made to comply with the Sponsor's request to collect blood from rats at various stages of the study and to perform analysis on the quantity of compound absorbed.

b. The second amendment does not apply to this phase of the study,

c. The third amendment was made to specify the final report date,

d. The fourth amendment was made to modifv the final report date, and

e. The fifth amendment was made to document that feed assays were performed by the Sponsor.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington Mass.
- Age at study initiation: weanling (35 days old)
- Housing: The animals in both phases of the study were individually housed in wire mesh suspended cages, except during breeding and weaning, when the dams and their offspring were kept in plastic cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period:Quarantinen for 14 day upon arrival.

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 73 + or - 2
- Humidity (%): 45% to 70% except during weekly hosing of tile floor for cleaning.
- Air changes (per hr): 10 times
Route of administration:
oral: feed
Vehicle:
other: First 20 days of Phase I of the study: Certified Rodent Chow, No. 5002 from Purina Company, St. Louis, Missouri.; May 27, 1980 until the end of the study: Charles River R-M-H 3200 Certified Meal, manufactured by Agway of Syracuse, N.Y.
Details on exposure:
The diets were prepared weekly by mixing the appropriate amount of UV-2908 with the appropriate amount of the basic diet with a Patterson-Kelley
2 cubic feet V-shaped mixer. Fresh diets were prepared weekly bassed on the feed consumption data obtained two weeks before. Feed consumption for each animal was measured weekly during the quarantine and for the next ten weeks on test during Phase 1. Feed and water was provided ad libitum.
Details on mating procedure:
Beginning the first day of the 11th week on test the rats were pair-mated within their respective groups. The males were rotated to the next female up on the 6th and 12th day of mating, with the last male in each group mated with the first female of the same group. The male rats were removed on the 18th day of
breeding and euthanized soon thereafter and the testes saved in 10% buffered formalin, in case it became necessary to perform histopathological examination on the reproductive organs of any of these rats. The females were allowed to deliver their young.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
The dose levels were set in advance by the Sponsor. Mixing Analyses: These analyses were performed by the Sponsor. Results sent to the lab indicated that the test article was properly mixed in the feed.
Duration of treatment / exposure:
18 Weeks
Frequency of treatment:
Daily thoroughly mixed in the feed.
Dose / conc.:
5 000 ppm
Remarks:
The low dose group received daily, thoroughly mixed in the feed, 5000 parts of UV-2908 per million parts of feed PPM corresponding to 0.5%.
Dose / conc.:
10 000 ppm
Remarks:
The mid-dose group received daily, thoroughly mixed in the feed, 10000 parts of UV-2908 per million parts of feed PPM corresponding to 1.0%.
Dose / conc.:
50 000 ppm
Remarks:
The high-dose group received daily, thoroughly mixed in the feed, 50000 parts of UV-2908 per million parts of feed PPM corresponding to 5%.
No. of animals per sex per dose:
High Dose: 20 female and 20 male
Mid Dose: 20 female and 20 male
Low Dose: 20 female and 20 male
Control: 20 female and 20 male
Control animals:
yes, plain diet
Positive control:
The control group of animals received the basic ration of feed for the duration of the study.
Parental animals: Observations and examinations:
All rats were inspected daily by the Animal Caretakers and/or by the study coordinators. Positive findings were documented as they occurred while negative findings were documented once a week. Any abnormal findings observed were immediately followed. Pertinent signs were called to the attention of the Study Director and all data were reviewed regularly by him.
Litter observations:
All rats were inspected daily by the Animal Caretakers and/or by the study coordinators. Positive findings were documented as they occurred while negative findings were documented once a week. Any abnormal findings observed were immediately followed. Pertinent signs were called to the attention of the Study Director and all data were reviewed regularly by him.
Postmortem examinations (parental animals):
The gonads were preserved in 10% buffered formalin in case it became necessary to perform histological examination of the reproductive organs of any of the rats.
Statistics:
The following determinations were statistically evaluated by PRL using the independent, two sided Students t-test at the 9S% confidence level:
a. Feed consumption data
b. Body weight data

Scheffe, H.: Analysis of Variance, Wiley &: Sons, New York, N.Y., 1959.

Snedecor, G.W. an. Cochran, W.G.: Statistical Analysis, 6th ed., Iowa State Univ. press, Ames, La, 1967.
Reproductive indices:
Fertility Index: No. of pregnancies - No. of matings; Viability Index: No. of live pups at 4 days - No. of liveborn pups; Lactation Index: No. of weaned pups - (No. of live pups at day 4 - No. of pups discarded at day 4); Gestation Index: No. of litters born alive - No. of females pregnant
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
The gonads were preserved in 10% buffered formalin in case it became necessary to perform histological examination of the reproductive organs of any of the rats.
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
It is clear that the reproductive process was very successful and that the compound at all dose levels appeared to have no detrimental effect.
Dose descriptor:
NOEL
Effect level:
50 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: All indices (fertility, viability, lactation, and gestation) were very high, indicating that the reproductive process was successful and that the compound at all dose levels did not influence the reproduction process.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
No abnormalities were observed during the course of breeding, hence, the authors made the conclusion that the compound did not influence reproduction/teratogenicity.
Dose descriptor:
NOEL
Generation:
F1
Effect level:
50 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on reproduction were seen at the highest dose level
Reproductive effects observed:
not specified
Conclusions:
All indices fertility, viability, lactation, and gestation were very high, indicating that the reproductive process was successful and that the compound at all dose levels did not influence the reproduction process.
Executive summary:

Reproductive process was successful and the compound at all dose levels did not influence the reproduction process. GLP compliance was followed. Test substance doses used in the study were as follows: low dose group received daily, thoroughly mixed in the feed, 5000 parts of UV-2908 per million parts of feed PPM 0.5%; mid-dose group received daily, thoroughly mixed in the feed, 10000 parts of UV-2908 per million parts of feed PPM 1.0%; high-dose group received daily, thoroughly mixed in the feed, 50000 parts of UV-2908 per million parts of feed PPM 5%.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Short description of key information:
All indices fertility, viability, lactation, and gestation were very high, indicating that the reproductive process was successful and that the compound at all dose levels did not influence the reproduction process. No abnormalities were observed during the course of breeding, hence, the authors made the conclusion that the compound did not influence reproduction/teratogenicity.

Effects on developmental toxicity

Description of key information

Under the experimental conditions of the study (OECD 414), 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.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
May 7, 1980 through September 12, 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Insufficient documentation of experimental methods and examination procedures. In general, this study was performed according to the SOPs, which are in conformity with the FDA GLP Regulations. Deviations were limited, listed below, and are believed not to impact the reliability of the test result.
Qualifier:
according to
Guideline:
other: Breeding/Reproductive
Deviations:
yes
Remarks:
See below
Principles of method if other than guideline:
a. The first amendment was made to comply with the Sponsor's request to collect blood from rats at various stages of the study and to perform analysis on the quantity of compound absorbed.

b. The second amendment does not apply to this phase of the study,

c. The third amendment was made to specify the final report date,

d. The fourth amendment was made to modifv the final report date, and

e. The fifth amendment was made to document that feed assays were performed by the Sponsor.
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington Mass.
- Housing: The animals in both phases of the study were individually housed in wire mesh suspended cages, except during breeding and weaning, when the dams and their offspring were kept in plastic cages.
- Diet: ad libitum
- Water: ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 73 + or - 2
- Humidity (%): 45% to 70% except during weekly hosing of tile floor for cleaning.
- Air changes (per hr): 10 times
Route of administration:
oral: feed
Vehicle:
other: First 20 days of Phase I of the study: Certified Rodent Chow, No. 5002 from Purina Company, St. Louis, Missouri.; May 27, 1980 until the end of the study: Charles River R-M-H 3200 Certified Meal, manufactured by Agway of Syracuse, N.Y.
Details on exposure:
The diets were prepared weekly by mixing the appropriate amount of UV-2908 with the appropriate amount of the basic diet with a Patterson-Kelley
2 cubic feet V-shaped mixer. Fresh diets were prepared weekly bassed on the feed consumption data obtained two weeks before. Feed consumption for each animal was measured weekly during the quarantine and for the next ten weeks on test during Phase 1. Feed and water was provided ad libitum.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
The dose levels were set in advance by the Sponsor. Mixing Analyses: These analyses were performed by the Sponsor. Results sent to the lab indicated that the test article was properly mixed in the feed.
Details on mating procedure:
Beginning the first day of the 11th week on test the rats were pair-mated within their respective groups. The males were rotated to the next female up on the 6th and 12th day of mating, with the last male in each group mated with the first female of the same group. The male rats were removed on the 18th day of breeding and euthanized soon thereafter and the testes saved in 10% buffered formalin, in case it became necessary to perform histopathological examination on the reproductive organs of any of these rats. The females were allowed to deliver their young.
Duration of treatment / exposure:
36 Days
Frequency of treatment:
Daily thoroughly mixed in the feed.
Duration of test:
36 Days
Control animals:
yes, plain diet
Maternal examinations:
All rats were inspected daily by the Animal Caretakers and/or by the study coordinators. Positive findings were documented as they occurred while negative findings were documented once a week. Any abnormal findings observed were immediately followed. Pertinent signs were called to the attention of the Study Director and all data were reviewed regularly by him.
Fetal examinations:
All rats were inspected daily by the Animal Caretakers and/or by the study coordinators. Positive findings were documented as they occurred while negative findings were documented once a week. Any abnormal findings observed were immediately followed. Pertinent signs were called to the attention of the Study Director and all data were reviewed regularly by him.
Statistics:
The following determinations were statistically evaluated by PRL using the independent, two sided Students t-test at the 9S% confidence level:
a. Feed consumption data
b. Body weight data

Scheffe, H.: Analysis of Variance, Wiley &: Sons, New York, N.Y., 1959.

Snedecor, G.W. an. Cochran, W.G.: Statistical Analysis, 6th ed., Iowa State Univ. press, Ames, La, 1967.
Indices:
fertility, viability, lactation, and gestation
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No abnormalities were observed during the course of breeding.
Dose descriptor:
NOEL
Effect level:
50 000 ppm
Based on:
other: The high-dose group received daily, thoroughly mixed in the feed, 50000 parts of UV-2908 per million parts of feed PPM 5%.
Basis for effect level:
other: effect type not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No abnormalities were observed during the course of breeding, hence, the authors made the conclusion that the compound did not influence reproduction/teratogenicity.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
No abnormalities were observed during the course of breeding, hence, the authors made the conclusion that the compound did not influence reproduction/teratogenicity.
Executive summary:

The study compound did not influence reproduction/teratogenicity. GLP compliance was followed. Test substance doses used in the study were as follows: low dose group received daily, thoroughly mixed in the feed, 5000 parts of UV-2908 per million parts of feed PPM 0.5%; mid-dose group received daily, thoroughly mixed in the feed, 10000 parts of UV-2908 per million parts of feed PPM 1.0%; high-dose group received daily, thoroughly mixed in the feed, 50000 parts of UV-2908 per million parts of feed PPM 5%.

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
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
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
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
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
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
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.
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
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
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.
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
Developmental effects observed:
no

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.

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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

As no adverse effects were observed in the developmental toxicity study (OECD 414) as well as in the supporting study (91-day treatment feeding study in the rat), the substance should not be classified for reprotoxicity under the criteria of the CLP GHS.